Use of Maleimide Derivatives for Preventing and Treating Cancer

ABSTRACT

The present invention is related to a compound of formula (I): 
     
       
         
         
             
             
         
       
     
     a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, a metabolite thereof or a prodrug thereof;
 
for use in a method for the treatment and/or prevention of cancer,
 
wherein
 
X is selected from the group consisting of N—R 1 , O and S;
 
R 1  is selected from the group consisting of alkyl, cycloalkyl, aryl, arylalkyl and hydrogen;
 
R 2  is selected from the group consisting of indolyl, substituted indolyl, azaindolyl and substituted azaindolyl; and
 
R 3  is selected from the group consisting of aryl, substituted aryl, unsubstituted heteroaryl, heterocyclyl and substituted heterocyclyl.

The present invention relates to a chemical compound of formula (I), itsuse in the treatment of a disease, a pharmaceutical compositioncomprising the compound, and a method for the treatment of a disease.

Cancer is the second leading cause of death in many countries, exceededonly by heart disease. Despite recent advances in cancer diagnosis andtreatment, surgery and radiotherapy may be curative if a cancer is foundearly, but current drug therapies for metastatic disease are mostlypalliative and seldom offer a long-term cure. Even with newchemotherapies entering the market, the need continues for new drugseffective in monotherapy or in combination with existing agents as firstline therapy, and as second and third line therapies in treatment ofresistant tumors.

Cancer therapy is a rapidly evolving field of science, nowadays growingmore and more into targeted therapies, employing antibodies to cancerspecific epitopes or small molecules interfering with cancer-specificpathways. Even in the face of these great progressions there is still ahigh clinical value and a strong need for innovations, due to betterunderstood resistances, high toxicities and adverse effects for drugsaffecting the microtubules in eukaryotic cells. Such microtubule-bindingagents can be classified by their affinity to different binding sites onmicrotubules and/or aB-tubulin heterodimers. These drugs impair thebalance of ‘dynamic instability’ in microtubules by either promotingtheir assembly and stability or by impairing the assembly of tubulinheterodimers, thus particularly interfering with cell progression duringmitosis, ultimately supporting induction of apoptosis (Matson andStukenberg, Spindle poisons and cell fate: a tale of two pathways. Mol.Interv. 11, 2011). Indoles from natural sources, as well assemi-synthetic and synthetic products have been described to displayanti-mitotic features due to inhibition of tubulin polymerization, mostof them by binding to the colchicine site (Brancaleand Silvestri,Indole, a core nucleus for potent inhibitors of tubulin polymerization.Med.Res.Rev. 27, 2007). Especially the tubulin-inhibiting agents havegiven rise to a set of small molecules, among them the combrestatins aslead substances, interfering with cancer vasculature, a promising targetin anticancer therapy (Kanthouand Tozer, Microtubule depolymerizingvascular disrupting agents: novel therapeutic agents for oncology andother pathologies. Int.J.Exp.Pathol. 90, 2009). Thus small moleculescontaining an indole as core structure are of special interest to beemployed in cancer treatment.

The problem underlying the present invention is the provision of a meanssuitable for the treatment of cancer. A further problem underlying thepresent invention is the provision of a pharmaceutical compositionsuitable for the treatment of cancer. A still further problem underlyingthe present invention is the provision of a method for the treatment ofcancer.

The problem underlying the present invention is solved by the subjectmatter of the attached independent claims, preferred embodiments may betaken from the attached dependent claims. Further aspects of theinvention and various embodiments thereof are disclosed in thefollowing.

Embodiment 1

A compound of formula (I):

a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvatethereof, a metabolite thereof or a prodrug thereof;

for use in a method for the treatment and/or prevention of cancer,

wherein

X is selected from the group consisting of N—R′, 0 and S;

R¹ is selected from the group consisting of alkyl, cycloalkyl, aryl,arylalkyl and hydrogen;

R² is selected from the group consisting of indolyl, substitutedindolyl, azaindolyl and substituted azaindolyl; and

R³ is selected from the group consisting of aryl, substituted aryl,unsubstituted heteroaryl, heterocyclyl and substituted heterocyclyl.

Embodiment 2

The compound of embodiment 1, wherein

R² comprises one, two, three, four, five or six substituents, wherebyeach and any of the substituents is individually and independentlyselected from the group comprising halogen, alkyl, alkenyl, alkynyl,acyl, formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio,arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl,alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro,alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, aryl sulfonyl,alkylsulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido.

Embodiment 3

The compound of any one of embodiments 1 to 2, wherein

R³ comprises one, two, three, four, five, six or seven substituents,whereby each and any of the substituents is individually andindependently selected from the group comprising hydrogen, alkyl,alkenyl, alkynyl, aryl, acyl, formyl, halogen, haloalkyl, alkylthio,monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, hydroxy, alkoxy, aryloxy, carboxyl, alkoxycarbonyl,aryloxycarbonyl, cyano, amino, amido, acylamino, nitro, alkylsulfinyl,aryl sulfinyl, alkyl sulfonyl, aryl sulfonyl, alkyl sulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido.

Embodiment 4

The compound of any one of embodiments 1 to 3, wherein

X is N—R¹, and wherein

R¹ is preferably selected from the group consisting of alkyl, hydrogen,phenyl and benzyl.

Embodiment 5

The compound of embodiment 4, wherein

R¹ is selected from the group consisting of methyl, butyl and hydrogen,preferably R¹ is selected from the group consisting of methyl andhydrogen.

Embodiment 6

The compound of any one of embodiments 1 to 3, wherein X is O.

Embodiment 7

The compound of any one of embodiments 1 to 6, preferably any one ofembodiments 4 to 6, wherein

R³ is selected from the group consisting of monocyclic aryl, substitutedmonocyclic aryl, bicyclic aryl, substituted bicyclic aryl, monocyclicheteroaryl, substituted monocyclic heteroaryl, bicyclic heteroaryl andsubstituted bicyclic heteroaryl.

Embodiment 8

The compound of embodiment 7, wherein

R³ is selected from the group consisting of phenyl, substituted phenyl,naphthenyl, substituted naphthenyl, heteroaryl with 5, 6, 9 or 10 ringatoms and substituted heteroaryl with 5, 6, 9 or 10 atoms, whereinheteroaryl contains 1 or 2 heteroatoms, wherein each and any of theheteroatoms is selected from the group consisting of N, O and S, whereinpreferably heteroaryl is selected from the group consisting of indolyl,thiophenyl and pyridinyl, and substituted heteroaryl is selected fromthe group consisting of substituted indolyl, substituted thiophenyl andsubstituted pyridinyl.

Embodiment 9

The compound of embodiment 8, wherein each and any of the substituentsis individually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 10

The compound of embodiment 9, wherein

R³ is selected from the group consisting of phenyl and substitutedphenyl, wherein substituted phenyl is phenyl consisting of one, two orthree substituents, wherein each and any of the substituents isindividually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 11

The compound of embodiment 8, wherein R³ is substituted phenyl and eachand any of the substituents is individually any independently selectedfrom the group consisting of alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkynly and halogen.

Embodiment 12

The compound of embodiment 11, wherein

alkyl is methyl or ethyl,

substituted alkyl is halogen-substituted methyl or acetyl,

alkoxy is ethoxy, and

alkynyl is vinyl.

Embodiment 13

The compound of embodiment 8, wherein the compound is of formula (IV)

wherein R⁵ is selected from the group consisting of alkyl, aminoalkyl,alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl.

Embodiment 14

The compound of embodiment 13, wherein

R⁵ is methyl.

Embodiment 15

The compound of any of embodiments 1 to 14, preferably any one ofembodiments 1 to 6, more preferably any one of embodiments 4 to 6,wherein each and any of indolyl, substituted indolyl, azaindolyl andsubstituted azaindolyl of R² is individually and independently eitherunprotected or protected at N, preferably at N of the 5-membered ring.

Embodiment 16

The compound of any one of embodiments 1 to 15, preferably any one ofembodiments 1 to 6, more preferably any one of embodiments 4 to 6,wherein

R² is a moiety of formula (VIa)

wherein

R⁴ is selected from the group consisting of hydrogen, aryl, heteroaryl,alkyl, cycloalkyl, polyfluoroalkyl, arylalkyl and heteroarylalkyl,

R⁶ is selected from the group consisting of alkyl and aryl,

each and any of Y¹, Y², Y³ and Y⁴ is individually and independentlyselected from the group consisting of N and CR⁷, under the proviso thatat least two of Y¹, Y², Y³ and Y⁴ are CR⁷,

wherein

each and any of R⁷ is individually and independently selected from thegroup consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, acyl,formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio,arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl,alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro,alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, arylsulfonyl,alkylsulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido,preferably each and any of R⁷ is individually and independently selectedfrom the group consisting of methyl and methoxy, more preferably R⁷ is5-methoxy or 5-halogen.

Embodiment 17

The compound of embodiment 16, wherein each and any of Y¹, Y², Y³ and Y⁴is CR⁷.

Embodiment 18

The compound of embodiment 17, wherein

R⁷ is hydrogen.

Embodiment 19

The compound of any one of embodiments 16 to 18, preferably of any oneof embodiments 17 to 18, wherein

R⁴ is selected from the group consisting of hydrogen, alkyl and benzyl,preferably R⁴ is hydrogen or methyl, more preferably R⁴ is hydrogen.

Embodiment 20

The compound of any one of embodiments 16 to 19, preferably any one ofembodiments 17 to 19, wherein

R6 is hydrogen or alkyl, preferably hydrogen or methyl, more preferablymethyl.

Embodiment 21

The compound of embodiment 16, wherein

each and any of Y¹, Y², Y³ and Y⁴ is individually and independentlyselected from the group consisting of N and CR⁷, under the proviso thatone or two of Y¹, Y², Y³ and Y⁴ are N.

Embodiment 22

The compound of embodiment 21, wherein

R⁷ is hydrogen.

Embodiment 23

The compound of any one of embodiments 16 and 21 to 22, preferably ofany one of embodiments 21 to 22, wherein

R⁴ is selected from the group consisting of hydrogen, alkyl and benzyl,preferably R⁴ is hydrogen or methyl, more preferably R⁴ is hydrogen.

Embodiment 24

The compound of any one of embodiments 16 and 21 to 23, preferably anyone of embodiments 21 to 23, wherein

R⁶ is hydrogen or alkyl, preferably hydrogen or methyl, more preferablymethyl.

Embodiment 25

The compound of any one of embodiments 1 to 24, preferably any one ofembodiments 16 to 24, wherein

R³ is selected from the group consisting of monocyclic aryl, substitutedmonocyclic aryl, bicyclic aryl, substituted bicyclic aryl, monocyclicheteroaryl, substituted monocyclic heteroaryl, bicyclic heteroaryl andsubstituted bicyclic heteroaryl.

Embodiment 26

The compound of embodiment 25, wherein

R³ is selected from the group consisting of phenyl, substituted phenyl,naphthenyl, substituted naphthenyl, heteroaryl with 5, 6, 9 or 10 ringatoms and substituted heteroaryl with 5, 6, 9 or 10 ring atoms, whereinheteroaryl contains 1 or 2 heteroatoms, wherein each and any of theheteroatoms is selected from the group consisting of N, O and S, whereinpreferably heteroaryl is selected from the group consisting of indolyl,thiophenyl and pyridinyl, and substituted heteroaryl is selected fromthe group consisting of substituted phenyl, substituted thiophenyl andsubstituted pyridinyl.

Embodiment 27

The compound of embodiment 26, wherein each and any of the substituentsis individually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 28

The compound of embodiment 27, wherein

R³ is selected from the group consisting of phenyl and substitutedphenyl, wherein substituted phenyl is phenyl consisting of one, two orthree substituents, wherein each and any of the substituents isindividually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 29

The compound of embodiment 26, wherein each and any of the substituentsis individually any independently selected from the group consisting ofalkyl, substituted alkyl, alkoxy, substituted alkoxy, alkynly andhalogen.

Embodiment 30

The compound of embodiment 29, wherein

alkyl is methyl or ethyl,

substituted alkyl is halogen-substituted methyl or acetyl,

alkoxy is ethoxy, and

alkynyl is vinyl.

Embodiment 31

The compound of any one of embodiments 16 to 26, preferably embodiment26, wherein the compound is of formula (VI)

wherein R⁵ is selected from the group consisting of alkyl, aminoalkyl,alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl.

Embodiment 32

The compound of any one of embodiments 16 to 26, preferably embodiment26, wherein the compound is of formula (V)

Embodiment 33

The compound of any one of embodiments 31 and 32, wherein

R⁵ is methyl.

Embodiment 34

The compound of any one of embodiments 1 to 33, wherein the compound isselected from the group consisting of1-Methyl-3,4-bis-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;

-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(4-vinylphenyl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione;-   3-(4-Acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione    (also referred to herein as PDA-66);-   3-(2,6-Dimethylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(3-Chlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(2,4-Dichlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(thiophen-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(pyridin-4-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione;-   3-(2,5-Dimethoxyphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(2-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione;-   3-(4-Fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(5-Acetyl-2-fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   N-(4-(1-Methyl-4-(2-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)    acetamide;-   3-(2-Methyl-1H-indol-3-yl)-4-phenylfuran-2,5-dione;-   3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)furan-2,5-dione;-   3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione;-   3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)furan-2,5-dione; and-   3-(2-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

Embodiment 35

The compound of any one of embodiments 1 to 34, wherein the compound is3-(4-acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Embodiment 36

The compound of any one of embodiments 1 to 34, wherein the compound is3-(4-acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Embodiment 37

A compound of formula (I):

a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvatethereof, a metabolite thereof or a prodrug thereof;

wherein

X is selected from the group consisting of N—R¹, O and S;

R¹ is selected from the group consisting of alkyl, cycloalkyl, aryl,arylalkyl and hydrogen;

R² is selected from the group consisting of indolyl, substitutedindolyl, azaindolyl and substituted azaindolyl; and

R³ is selected from the group consisting of aryl, substituted aryl,unsubstituted heteroaryl, heterocyclyl and substituted heterocyclyl.

Embodiment 38

The compound of embodiment 37, wherein

R² comprises one, two, three, four, five or six substituents, wherebyeach and any of the substituents is individually and independentlyselected from the group comprising halogen, alkyl, alkenyl, alkynyl,acyl, formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio,arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl,alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro,alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, aryl sulfonyl,alkylsulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido.

Embodiment 39

The compound of any one of embodiments 37 to 38, wherein

R³ comprises one, two, three, four, five, six or seven substituents,whereby each and any of the substituents is individually andindependently selected from the group comprising hydrogen, alkyl,alkenyl, alkynyl, aryl, acyl, formyl, halogen, haloalkyl, alkylthio,monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, hydroxy, alkoxy, aryloxy, carboxyl, alkoxycarbonyl,aryloxycarbonyl, cyano, amino, amido, acylamino, nitro, alkylsulfinyl,arylsulfinyl, alkyl sulfonyl, aryl sulfonyl, alkylsulfinamido,arylsulfinamido, alkylsulfonamido and arylsulfonamido.

Embodiment 40

The compound of any one of embodiments 37-39, wherein

X is N—R¹, and wherein

R¹ is preferably selected from the group consisting of alkyl, hydrogen,phenyl and benzyl.

Embodiment 41

The compound of embodiment 40, wherein

R¹ is selected from the group consisting of methyl, butyl and hydrogen,preferably R¹ is selected from the group consisting of methyl andhydrogen.

Embodiment 42

The compound of any one of embodiments 37 to 39, wherein X is O.

Embodiment 43

The compound of any one of embodiments 37 to 42, preferably any one ofembodiments 4 to 6, wherein

R³ is selected from the group consisting of monocyclic aryl, substitutedmonocyclic aryl, bicyclic aryl, substituted bicyclic aryl, monocyclicheteroaryl, substituted monocyclic heteroaryl, bicyclic heteroaryl andsubstituted bicyclic heteroaryl.

Embodiment 44

The compound of embodiment 43, wherein

R³ is selected from the group consisting of phenyl, substituted phenyl,naphthenyl, substituted naphthenyl, heteroaryl with 5, 6, 9 or 10 ringatoms and substituted heteroaryl with 5, 6, 9 or 10 atoms, whereinheteroaryl contains 1 or 2 heteroatoms, wherein each and any of theheteroatoms is selected from the group consisting of N, O and S, whereinpreferably heteroaryl is selected from the group consisting of indolyl,thiophenyl and pyridinyl, and substituted heteroaryl is selected fromthe group consisting of substituted indolyl, substituted thiophenyl andsubstituted pyridinyl.

Embodiment 45

The compound of embodiment 44, wherein each and any of the substituentsis individually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 46

The compound of embodiment 45, wherein

R³ is selected from the group consisting of phenyl and substitutedphenyl, wherein substituted phenyl is phenyl consisting of one, two orthree substituents, wherein each and any of the substituents isindividually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 47

The compound of embodiment 44, wherein R³ is substituted phenyl and eachand any of the substituents is individually any independently selectedfrom the group consisting of alkyl, substituted alkyl, alkoxy,substituted alkoxy, alkynly and halogen.

Embodiment 48

The compound of embodiment 47, wherein

alkyl is methyl or ethyl,

substituted alkyl is halogen-substituted methyl or acetyl,

alkoxy is ethoxy, and

alkynyl is vinyl.

Embodiment 49

The compound of embodiment 44, wherein the compound is of formula (IV)

wherein R⁵ is selected from the group consisting of alkyl, aminoalkyl,alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl.

Embodiment 50

The compound of embodiment 49, wherein

R⁵ is methyl.

Embodiment 51

The compound of any of embodiments 37 to 50, preferably any one ofembodiments 1 to 6, more preferably any one of embodiments 4 to 6,wherein each and any of indolyl, substituted indolyl, azaindolyl andsubstituted azaindolyl of R² is individually and independently eitherunprotected or protected at N, preferably at N of the 5-membered ring.

Embodiment 52

The compound of any one of embodiments 37 to 51, preferably any one ofembodiments 37 to 42, more preferably any one of embodiments 40 to 42,wherein

R² is a moiety of formula (VIa)

wherein

R⁴ is selected from the group consisting of hydrogen, aryl, heteroaryl,alkyl, cycloalkyl, polyfluoroalkyl, arylalkyl and heteroarylalkyl,

R⁶ is selected from the group consisting of alkyl and aryl, each and anyof Y¹, Y², Y³ and Y⁴ is individually and independently selected from thegroup consisting of N and CR⁷, under the proviso that at least two ofY¹, Y², Y³ and Y⁴ are CR⁷,

wherein

each and any of R⁷ is individually and independently selected from thegroup consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, acyl,formyl, cycloalkyl, aryl, haloalkyl, polyfluoroalkyl, alkylthio,arylthio, monoalkylamino, dialkylamino, monoarylamino, diarylamino,alkylarylamino, alkylimido, hydroxy, alkoxy, aryloxy, carboxyl,alkoxycarbonyl, aryloxycarbonyl, cyano, amino, amido, acylamino, nitro,alkylsulfinyl, arylsulfinyl, alkyl sulfonyl, arylsulfonyl,alkylsulfinamido, arylsulfinamido, alkylsulfonamido and arylsulfonamido,preferably each and any of R⁷ is individually and independently selectedfrom the group consisting of methyl and methoxy, more preferably R⁷ is5-methoxy or 5-halogen.

Embodiment 53

The compound of embodiment 52, wherein each and any of Y¹, Y², Y³ and Y⁴is CR⁷.

Embodiment 54

The compound of embodiment 53, wherein

R⁷ is hydrogen.

Embodiment 55

The compound of any one of embodiments 52 to 54, preferably of any oneof embodiments 53 to 54, wherein

R⁴ is selected from the group consisting of hydrogen, alkyl and benzyl,preferably R⁴ is hydrogen or methyl, more preferably R⁴ is hydrogen.

Embodiment 56

The compound of any one of embodiments 52 to 55, preferably any one ofembodiments 53 to 55, wherein

R6 is hydrogen or alkyl, preferably hydrogen or methyl, more preferablymethyl.

Embodiment 57

The compound of embodiment 52, wherein each and any of Y¹, Y², Y³ and Y⁴is individually and independently selected from the group consisting ofN and CR⁷, under the proviso that one or two of Y¹, Y², Y³ and Y⁴ are N.

Embodiment 58

The compound of embodiment 57, wherein

R⁷ is hydrogen.

Embodiment 59

The compound of any one of embodiments 52 and 57 to 58, preferably ofany one of embodiments 57 to 58, wherein

R⁴ is selected from the group consisting of hydrogen, alkyl and benzyl,preferably R⁴ is hydrogen or methyl, more preferably R⁴ is hydrogen.

Embodiment 60

The compound of any one of embodiments 52 and 57 to 59, preferably anyone of embodiments 57 to 59, wherein

R⁶ is hydrogen or alkyl, preferably hydrogen or methyl, more preferablymethyl.

Embodiment 61

The compound of any one of embodiments 37 to 60, preferably any one ofembodiments 52 to 60, wherein

R³ is selected from the group consisting of monocyclic aryl, substitutedmonocyclic aryl, bicyclic aryl, substituted bicyclic aryl, monocyclicheteroaryl, substituted monocyclic heteroaryl, bicyclic heteroaryl andsubstituted bicyclic heteroaryl.

Embodiment 62

The compound of embodiment 61, wherein

R³ is selected from the group consisting of phenyl, substituted phenyl,naphthenyl, substituted naphthenyl, heteroaryl with 5, 6, 9 or 10 ringatoms and substituted heteroaryl with 5, 6, 9 or 10 ring atoms, whereinheteroaryl contains 1 or 2 heteroatoms, wherein each and any of theheteroatoms is selected from the group consisting of N, O and S, whereinpreferably heteroaryl is selected from the group consisting of indolyl,thiophenyl and pyridinyl, and substituted heteroaryl is selected fromthe group consisting of substituted phenyl, substituted thiophenyl andsubstituted pyridinyl.

Embodiment 63

The compound of embodiment 62, wherein each and any of the substituentsis individually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 64

The compound of embodiment 63, wherein

R³ is selected from the group consisting of phenyl and substitutedphenyl, wherein substituted phenyl is phenyl consisting of one, two orthree substituents, wherein each and any of the substituents isindividually and independently selected from the group consisting offluoro, chloro, methyl, trifluoromethyl, vinyl, acetyl, acetamido,methoxy, formyl, ethoxycarbonyl and ydimethylamidocarbonyl.

Embodiment 65

The compound of embodiment 62, wherein each and any of the substituentsis individually any independently selected from the group consisting ofalkyl, substituted alkyl, alkoxy, substituted alkoxy, alkynly andhalogen.

Embodiment 66

The compound of embodiment 65, wherein

alkyl is methyl or ethyl,

substituted alkyl is halogen-substituted methyl or acetyl,

alkoxy is ethoxy, and

alkynyl is vinyl.

Embodiment 67

The compound of any one of embodiments 52 to 62, preferably embodiment62, wherein the compound is of formula (VI)

wherein R⁵ is selected from the group consisting of alkyl, aminoalkyl,alkoxyalkyl, hydroxyalkyl, aryl and heteroaryl.

Embodiment 68

The compound of any one of embodiments 52 to 62, preferably embodiment62, wherein the compound is of formula (V)

Embodiment 69

The compound of any one of embodiments 67 and 68, wherein

R⁵ is methyl.

Embodiment 70

The compound of any one of embodiments 37 to 69, wherein the compound isselected from the group consisting of1-Methyl-3,4-bis-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;

-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(4-vinylphenyl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione;    3-(4-Acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione    (also referred to herein as PDA-66);-   3-(2,6-Dimethylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(3-Chlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(2,4-Dichlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(thiophen-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(pyridin-4-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione;-   3-(2,5-Dimethoxyphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   1-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(2-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione;-   3-(4-Fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(5-Acetyl-2-fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   N-(4-(1-Methyl-4-(2-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)    acetamide;-   3-(2-Methyl-1H-indol-3-yl)-4-phenylfuran-2,5-dione;-   3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione;-   3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)furan-2,5-dione;-   3-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione;-   3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)furan-2,5-dione; and-   3-(2-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

Embodiment 71

The compound of any one of embodiments 37 to 70, wherein the compound is3-(4-acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Embodiment 72

The compound of any one of embodiments 37 to 70, wherein the compound is3-(4-acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Embodiment 73

The compound of any one of embodiments 37 to 72, wherein R3 is differentfrom indolyl and/or substituted indolyl.

Embodiment 74

The compound of any one of embodiments 1 to 36, wherein cancer isselected from the group comprising breast cancer, lung cancer, includingnon-small cell lung carcinoma, renal cancer, colon cancer,myelodysplastic syndrome, genitourinary cancer, gastrointestinal cancer,epidermoid cancer, melanoma, glioma, ovarian cancer, pancreatic cancer,lymphoma, myeloma, colorectal cancer, neuroblastoma, head and/or neckcancer, bladder cancer, brain cancer in a broader sense and gastriccancer in a broader sense, any metastases of any thereof.

Embodiment 75

The compound of any one of embodiments 1 to 36, wherein cancer is breastcancer and metastases.

Embodiment 76

The compound of any one of embodiments 1 to 36, wherein cancer is lungcancer, including non-small cell lung cancer, and metastases thereof.

Embodiment 77

The compound of any one of embodiments 1 to 36, wherein cancer is renalcancer and metastases thereof.

Embodiment 78

The compound of any one of embodiments 1 to 36, wherein cancer is coloncancer and metastases thereof.

Embodiment 79

The compound of any one of embodiments 1 to 36, wherein cancer ismyelodysplastic syndrome and metastases thereof.

Embodiment 80

The compound of any one of embodiments 1 to 36, wherein genitourinarycancer and metastases thereof.

Embodiment 81

The compound of any one of embodiments 1 to 36, wherein cancer isgastrointestinal cancer and metastases thereof.

Embodiment 82

The compound of any one of embodiments 1 to 36, wherein cancer isepidermoid cancer and metastases thereof.

Embodiment 83

The compound of any one of embodiments 1 to 36, wherein cancer ismelanoma and metastases thereof.

Embodiment 84

The compound of any one of embodiments 1 to 36, wherein cancer is gliomaand metastases thereof.

Embodiment 85

The compound of any one of embodiments 1 to 36, wherein cancer isovarian cancer and metastases thereof.

Embodiment 86

The compound of any one of embodiments 1 to 36, wherein cancer ispancreatic cancer and metastases thereof.

Embodiment 87

The compound of any one of embodiments 1 to 36, wherein cancer islymphoma and metastases thereof.

Embodiment 88

The compound of any one of embodiments 1 to 36, wherein cancer ismyeloma and metastases thereof.

Embodiment 89

The compound of any one of embodiments 1 to 36, wherein cancer iscolorectal cancer and metastases and thereof.

Embodiment 90

The compound of any one of embodiments 1 to 36, wherein cancer isneuroblastoma and metastases thereof.

Embodiment 91

The compound of any one of embodiments 1 to 36, wherein cancer is headand/or neck cancer and metastases thereof.

Embodiment 92

The compound of any one of embodiments 1 to 36, wherein cancer isbladder cancer and metastases thereof.

Embodiment 93

The compound of any one of embodiments 1 to 36, wherein cancer is braincancer and metastases thereof.

Embodiment 94

The compound of any one of embodiments 1 to 36, wherein cancer isgastric neck cancer and metastases thereof.

Embodiment 95

The compound of any one of embodiments 1 to 36 and 74 to 94, wherein themethod comprises the administration of a second therapeutic agent,wherein the second therapeutic agent is a chemotherapeutic agent.

Embodiment 96

The compound of embodiment 95, wherein the chemotherapeutic agent isselected from the group comprising cytarabine, etoposide, mitoxantron,cyclophosphamide, retinoic acid, daunorubicin, doxorubicin, idarubicin,azacytidine, decitabine, a tyrosin-kinase inhibitor, a antineoplasticantibody, vincaalkaloids and steroids.

Embodiment 97

The compound of embodiment 96, wherein the chemotherapeutic agent is atyrosin-kinas inhibitor, wherein the tyrosin-kinase inhibitor isselected from the group comprising sorafenib, dasatinib, nilotinib,nelarabine and fludarabine.

Embodiment 98

The compound of embodiment 96, wherein the chemotherapeutic agent isAlemtuzumab (Campath®)

Embodiment 99

Use of compounds according to any one of embodiments 1 to 73 for themanufacture of a medicament against cancer.

Embodiment 100

A pharmaceutical compositions comprising a compound of any one ofembodiments 1 to 73 and a pharmaceutically acceptable carrier orexcipient.

Embodiment 101

The pharmaceutical composition of embodiment 100, wherein thepharmaceutical composition comprises a second therapeutic agent, whereinthe second therapeutic agent is a chemotherapeutic agent.

Embodiment 102

A method of treatment and/or prevention of cancer, wherein the methodcomprises administering to a subject in need thereof an therapeuticallyeffective amount of a compound of any one of embodiments 1 to 73 or of apharmaceutical composition of any one of embodiments 100 to 101.

The present invention is based on the surprising finding that that thecompound of the invention is capable of inhibition GSK3ß. Morespecifically, the present invention is based on the surprising findingthat the compound of the invention is suitable for the treatment ofcancer. Based on this finding, the compound of the invention may be usedin the treatment of any disease involving GSK3ß. In an embodiment canceris such disease involving GSK3ß, whereby cancer is preferably is any oneof breast cancer, lung cancer, including non-small cell lung carcinoma,renal cancer, colon cancer, myelodysplastic syndrome, genitourinarycancer, gastrointestinal cancer, epidermoid cancer, melanoma, glioma,ovarian cancer, pancreatic cancer, lymphoma, myeloma, colorectal cancer,neuroblastoma, head and/or neck cancer, bladder cancer, brain cancer ina broader sense and gastric cancer in a broader sense.

In an embodiment of the compound of the invention the indolyl,substituted indolyl, azaindolyl and substituted azaindolyl are each andindividually attached to the maleimide moiety via a 3′-position of theindolyl and azaindolyl, respectively.

In an embodiment of the invention the indolyl, substituted indolyl,azaindolyl and substituted azaindolyl are each and individually attachedto the maleimide moiety via a 3′-position of the indolyl and azaindolyl,respectively,

A compound of the invention may exist in free or in salt form and/orsolvate form or of the salt thereof. A “pharmaceutically acceptablesalt” of a compound relates to a salt that is pharmaceuticallyacceptable and that possesses the desired pharmacological activity ofthe parent compound. “Physiologically or pharmaceutically acceptablesalts” of a compounds of the invention include but are not limited toacid addition salts with a) inorganic acids, such as, for example,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid orphosphoric acid and the like, or formed with b) organic acids, includingbut not limited to carboxylic acids, such as, e.g., acetic acid,tartaric acid, lactic acid, citric acid, maleic acid, malonic acid,succinic acid, ascorbic acid, fumaric acid, cinnamic acid, mandelicacid, benzoic acid, gluconic acid and the like, or c) sulfonic acids,such as, e.g., methane sulfonic acid, benzene sulfonic acid, toluenesulfonic acid, camphorsulfonic acid and the like.

Physiologically acceptable solvates are preferably hydrates.

Unless otherwise stated, the following terms used in the specificationand claims have, in a preferred embodiment, the meanings given below:

The terms “alkyl” and “alkyloxy” as preferably used herein or incombination with other terms means linear or branched hydrocarbonstructures and combinations thereof with one, two, three, four, five orsix carbon atoms, including but not limited to, e. g., methyl, ethyl,propyol (iso-, n-), butyl (iso-, n-, tert-), pentyl, hexyl, methoxy,ethoxy, propoxy (iso-, n-), butoxy (iso-, n-, tert-), pentoxy, hexoxyand the like.

As preferably used herein the term “cycloalkyl” means mono- orpolycyclic saturated or unsaturated three, four, five, six or seven ringcarbocyclic alkyl groups, including but not limited to cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclobutenyl,cyclopentenyl, cyclopentadienyl, cyclohexyl, cyclohexenyl,cyclohexadienyl, cycloheptenyl, cycloheptadienyl and cycloheptatrienyland the like.

The term “aryl” as preferably used herein means mono- and polycyclicaromatic groups having 6, 7, 8, 9, 10, 11, 12, 13 or 14 backbone carbonatoms, optionally fused to a carbocyclic group, including but notlimited to phenyl, naphthyl, indenyl, indanyl, azulenyl, fluorenyl,1,2,3,4-tetrahydronaphthyl, phenanthrenyl and the like.

The term “monoalkylamino” or “monoarylamino” as preferably used hereinmeans a radical —NHR where R is an alkyl, cycloalkyl or aryl as definedherein, including but not limited to, e. g., methylamino,cyclohexylamino, phenylamino and the like.

The term “dialkylamino” or “diarylamino” as preferably used herein meansa radical —NRR′, where each of R and R′ individually and independentlyrepresents an alkyl, cycloalkyl or aryl as defined herein, including butnot limited to, e. g., dimethylamino, dicyclohexylamino,methylethylamino, diphenylamino and the like.

The term “alkylthio” or “arylthio” as preferably used herein means aradical —SR where R is an alkyl or aryl as defined herein, including butnot limited to, e. g., methylthio, ethylthio, propylthio, butylthio,phenylthio and the like.

The term “acylamino” as preferably used herein means a radical-NR′C(O)R,where R′ is hydrogen or alkyl, and R is hydrogen, alkyl, cycloalkyl,cycloalkylalkyl, phenyl or phenylalkyl, wherein alkyl, cycloalkyl,cycloalkylalkyl, and phenylalkyl are as defined herein. Representativeexamples include, but are not limited to, formylamino, acetylamino,cylcohexylcarbonylamino, benzoylamino and the like.

The term “haloalkyl” as preferably used herein means substituted alkylas defined herein, wherein alkyl is substituted with one or more of sameor different halogen atoms, including but not limited to, e. g., —CH₂CI,—CF₃, —CH₂CF₃, —CH₂CCl₃ and the like.

The terms “alkylsufinyl” and “arylsulfinyl” as preferably used hereinmean a —S(O)R group, where R is alkyl (in case of alkylsulfinyl) andaryl (in case of arylsulfinyl) as defined herein, including but notlimited to, e.g., methylsulfinyl, ethylsulfinyl, propylsulfinyl,butylsulfinyl, each including all isomeric forms thereof, and the like.

The terms “alkylsulfonyl” and “arylsulfonyl” as preferably used hereinmean a —S(O)₂R group, where R is alkyl (in case of alkylsulfonyl) andaryl (in case of arylsulfonyl) as defined herein, including but notlimited to, e.g., methylsulfonyl, ethylsulfonyl, propylsulfonyl,butylsulfonyl, each including all isomeric forms thereof, and the like.

The terms “alkylsulfinamido” and arylsulfinamido” as preferably usedherein mean a —S(O)NRR′ group, where R and R′ are hydrogen and/or alkyl(in case of alkylsulfinamido) and aryl (in case of arylsulfinamido) asdefined herein, including but not limited to, e.g.,tert-butanesulfinamide, p-toluenesulfinamide and the like.

The terms “alkylsulfonamido” and arylsulfonamido” as preferably usedherein mean a —S(O)₂NRR′ group, where R and R′ are hydrogen and/or alkyl(in case of alkylsulfonamido) and aryl (in case of arylsulfonamido) asdefined herein, including but not limited to, e.g., methansulfonamideand the like.

The term “heteroaryl” as preferably used herein means mono- orbi-carbocyclic aromatic groups with 1, 2, 3 or 4 ring-heteroatomsselected from N, S and O. Preferably, a total number of ring atoms is 5,6, 7, 8, 9 or 10. Examples without limitation of heteroaryl groups arebenzofuranyl, furyl, thienyl, benzothienyl, thiazolyl, imidazolyl,oxazolyl, oxadiazolyl, thiadiazolyl, benzothiazolyl, triazolyl,tetrazolyl, isoxazolyl, isothiazolyl, pyrrolyl, pyranyl,tetrahydropyranyl, pyrazolyl, pyridyl, pyrimidinyl, quinolinyl,isoquinolynyl, purinyl, carbazolyl, benzoxazolyl, benzamidazolyl,indolyl, isoindolyl, diazinyl, pyrazinyl, triazinyl, tetrazinyl,tetrazolyl, benzothiophenyl, benzopyridyl, benzimidazolyl andderivatives thereof. The heteroaryl ring is optionally substitutedindependently with one or more substituents, wherein each and anysubstituent is individually and independently selected from alkyl,haloalkyl, heteroalkyl, alkoxy, hydroxy, halogen, nitro, cyano groupsand the like, preferably as defined herein.

The term “heterocyclyl” as preferably used herein means a mono- orpolycyclic saturated or unsaturated non-aromatic heterocyclyl groups of5, 6, 7 or 8 ring atoms in which one or two ring atoms are heteroatomsselected from NR (where R is independently hydrogen or alkyl, preferablyas defined herein), O, or S(O)_(n) (where n is an integer from 0, 1 and2), the remaining ring atoms being carbon atoms, where one or two carbonatoms may optionally be replaced by a carbonyl group. The heterocyclylring may be optionally substituted independently with one, two or threesubstituents, wherein each substituent is individually and independentlyselected from alkyl, haloalkyl, heteroalkyl, halogen, nitro, cyano,hydroxy, alkoxy, amino, mono- or dialkylamino, acyl, preferably asdefined herein. Examples for heterocyclyl groups include but are notlimited to tetrahydrofuranyl, tetrahydropyranyl, imidazolinyl,imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl,thiomorpholinyl, N-methylpiperidin-3-yl, N-methylpyrrolidin-3-yl,pyrrolinyl and derivatives of each thereof.

The term “halogen” as preferably used herein means a halogen atomselected from fluorine, chlorine, bromine and iodine, preferably thehalogen atom is either fluorine or chlorine, more preferably the halogenatom is fluorine.

The term “protected” as preferably used herein means those organicgroups intended to protect nitrogen atoms against undesirable reactionsduring synthetic procedures. Suitable nitrogen protecting groups arewell known in the art and include but are not limited to, e. g.,trimethylsilyl, tert-butyldimethylsilyl (TBDMS), benzyl,benzyloxycarbonyl (Cbz), 9-fluorenylmethoxycarbonyl (Fmoc),tert-butoxycrbonyl (Boc), trifluoroacetyl,2-trimethylsilylethanesulfonyl (SES), and the like. Other suitablenitrogen protecting groups which are suitable for the practicing of theinvention can be found in the publication of T. W. Greene and G. M.Wuts, “Protecting Groups in Organic Synthesis”, Second Edition, Wiley,New York, 1991, and references cited therein.

In an embodiment and as preferably used herein, a disease involvingGSK3B is a disease where cells expressing GSK3B and tissue expressingGSK3B, respectively, are either a or the cause for the disease and/orthe symptoms of the disease, or are part of the pathology underlying thedisease. In an embodiment of the disease, preferably when used inconnection with the treatment, treating and/or therapy of the disease,affecting the cells, the tissue and pathology, respectively, results incure, treatment or amelioration of the disease and/or the symptoms ofthe disease.

Numerous additional aspects and advantages of the invention will becomeapparent to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presentlypreferred embodiments thereof.

The invention also relates to the metabolites and prodrugs of thecompound of the invention. Preferably, a prodrug of a compound of theinvention is prepared by modifying functional groups present in thecompound of the invention in such a way that the modifications may becleaved in vivo to release a or the active compound. Preferably, suchactive compound is a compound of the invention or a compound derivedtherefrom having at least one characteristic of a compound of theinvention. Preferably, such characteristic is the capacity to inhibitGSK3ß and/or the suitability for the treatment of a GKS3b involvingdisease, preferably cancer.

In accordance therewith the term “prodrug” refers to (a) an inactiveform of a drug that exerts its effects after metabolic processes invivo, when such prodrug is administered to a mammalian subject, torelease an active parent drug and preferably a compound of theinvention, or (b) a substance that gives rise to a pharmacologicallyactive metabolite, although not itself active (i.e. an inactiveprecursor). Examples of prodrugs include, but are not limited to esters,carbamates and the like.

As preferably used herein the term “metabolite” refers to a) a productof metabolism, including an intermediate and an end product, b) anysubstance in metabolism (either as product of metabolism or as necessaryfor metabolism), or c) any substance produced or used during metabolism.More preferably, the term “metabolite” refers to an end product thatremains after metabolism.

As preferably used herein, the term “pharmaceutically acceptableexcipient” an excipient that is useful in preparing a pharmaceuticalcomposition that is generally safe, non-toxic and neither biologicallynor otherwise undesirable, or adversely affects the therapeutic benefitof the compound of the invention. A “pharmaceutically acceptableexcipient” as preferably used in the specification and claims includesboth one and more than one such excipient. Such excipient may be anysolid, liquid, semi-solid. Solid pharmaceutical excipients includestarch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice,flour, chalk, silica gel, magnesium stearate, sodium stearate, glycerolmonostearate, sodium chloride, dried skim milk and the like. Liquid andsemisolid excipients may be selected from glycerol, propylene glycol,water, ethanol and various oils, including those of petroleum, animal,vegetable or synthetic origin, e. g., peanut oil, soybean oil, mineraloil, sesame oil, and the like.

As preferably used herein, the term “therapeutically effective amount”means the amount of a compound of the invention formula (I) that, whenadministered to a mammal for treating a disease, is sufficient to effectsuch treatment for the disease. The “therapeutically effective amount”will vary depending on the compound, the disease and its severity andthe age, weight, etc., of the mammal to be treated.

As preferably used herein, “treating” or “treatment” of a diseaseincludes: (1) preventing the disease, i. e. causing the clinicalsymptoms of the disease not to develop in a mammal that may be exposedto or predisposed to the disease but does not yet experience or displaysymptoms of the disease, (2) inhibiting the disease, i. e., arresting orreducing the development of the disease or its clinical symptoms, or (3)relieving the disease, i. e., causing regression of the disease or itsclinical symptoms.

The term “treatment of cancer” includes partial or total inhibition ofcancer in a subject, as well as partial or total destruction of thecancer cells.

As preferably used herein, the term “prevention of cancer” includespreventing the onset of clinically evident cancer as well as preventingthe onset of a preclinical evident stage of cancer in subjects at risk.

In an embodiment of the invention, cancer is a solid cancer andmetastases thereof. In a preferred embodiment, cancer is selected fromthe group comprising breast cancer, lung cancer, including non-smallcell lung carcinoma, renal cancer, colon cancer, myelodysplasticsyndrome, genitourinary cancer, gastrointestinal cancer, epidermoidcancer, melanoma, glioma, ovarian cancer, pancreatic cancer, lymphoma,myeloma, colorectal cancer, neuroblastoma, head and/or neck cancer,bladder cancer, brain cancer in a broader sense and gastric cancer in abroader sense, including metastases of any one thereof.

In an embodiment of the invention, cancer is resistant cancer and inparticular multidrug resistant cancer, i.e., the cancer cells exhibitresistance to conventional chemotherapeutics, preferably the MDR(multidrug resistance) phenotype; preferably, such resistant cancer isselected from the group comprising breast cancer, lung cancer, includingnon-small cell lung carcinoma, renal cancer, colon cancer,myelodysplastic syndrome, genitourinary cancer, gastrointestinal cancer,epidermoid cancer, melanoma, glioma, ovarian cancer, pancreatic cancer,lymphoma, myeloma, colorectal cancer, neuroblastoma, head and/or neckcancer, bladder cancer, brain cancer in a broader sense and gastriccancer in a broader sense.

In an embodiment, the compound of the invention is a compound, aphysiologically acceptable salt thereof or a physiologically acceptablesolvate thereof, which is capable of stimulating apoptosis in cancercells.

The present invention thus also relates to the use of a compound of theinvention, a physiologically acceptable salt or solvate thereof,preferably as defined herein, in combination with one or more than onefurther chemotherapeutic agent.

In an embodiment of the invention, the treatment of the subjectcomprises further stimulation of cell death by a conventional method orcombination of conventional methods. The conventional methods preferablybeing selected from the group consisting of irradiation, e. g. externalirradiation or administration of radioactive compounds, bone marrowtransplantation and treatment with a chemotherapeutic agent which isincluding antineoplastic agents, multidrug resistance reversing agents,and biological response modifiers, and combinations thereof.

The present invention thus also relates to the use of a compound of theinvention, a physiologically acceptable salt or a solvate thereof,preferably as defined herein, in combination with one or more than onefurther chemotherapeutic agent. Suitable antineoplastic agents may beselected from the group comprising asparaginase, bleomycin, busulfan,carmustine, chlorambucil, cladribine, cyclophosphamide, cytarabine,dacarbazine, daunorubicin, doxorubicin, etoposide, fludarabine,gemcitabine, hydroxyurea, idarubicin, ifosfamide, lomustine,mechlorethamine, melphalan, mercaptopurine, methotrexate, mitomycin,mitoxantrone, pentostatin, procarbazine, 6-thioguanine, topotecan,vinblastine, vincristine, dexamethasone, retinoic acid and prednisone.Preferred examples for antineoplastic agents to be used in the treatmentof cancer in accordance with the present invention, especially in thetreatment of AML, or ALL, comprise cytarabine, etoposide, mitoxantron,cyclophosphamide, retinoic acid, daunorubicin, doxorubicin andidarubicin.

When the compounds of formula (I), physiologically acceptable salts orsolvates thereof are to be used as active ingredients in the uses,methods and compositions of the present invention, they can beincorporated into standard pharmaceutical dosage forms, which theskilled artisan is familiar with. Basically, any pharmaceutical dosageform may be used in the invention.

The present invention thus also relates to a pharmaceutical compositioncomprising a pharmaceutically acceptable auxiliary agent in addition toa compound of the invention, a physiologically acceptable salt orsolvate thereof as defined above. Such auxiliary agents are known in theart. e. g., the usual pharmaceutical excipients, diluents and adjuvants,e.g., organic and inorganic inert carrier materials such as water,gelatine, lactose, starch, magnesium stearate, talc, vegetable oils,gums, polyalkylene glycols, etc. These pharmaceutical preparations canbe employed in a solid form, e.g., as tablets, capsules, or they can beadministered in liquid form, e.g., as solutions, suspensions oremulsions.

Further pharmaceutical excipients and adjuvants which may be added to apharmaceutical composition, include preservatives, antioxidants,antimicrobial agents and other stabilizers; wetting, emulsifying andsuspending agents, and anti-caking compounds; fragrance and coloringadditives; compositions for improving compressibility, or agents tocreate a delayed, sustained or controlled release of the activeingredient; and various salts to change the osmotic pressure of thepharmaceutical preparation or to act as buffers. Such excipients andadjuvants are known to the skilled artisan.

It will be acknowledged by a person skilled in the art that a or thecompound of the invention is any compound disclosed herein, includingbut not limited to any compound described in any of the aboveembodiments and any of the following embodiments.

It will be acknowledged by a person skilled in the art that a or themethod of the invention is any method disclosed herein, including butnot limited to any method described in any of the above embodiments andany of the following embodiments.

It will be acknowledged by a person skilled in the art that a or thecomposition of the invention is any composition disclosed herein,including but not limited to any composition described in any of theabove embodiments and any of the following embodiments.

As to the synthesis of the compound of the invention a person skilled inthe art will acknowledge the following. Disubstituted maleimide andparticularly bisindolylmaleimide subunit is present in a number ofbiologically active compounds. Among these arcyriarubins (Scheme 1; a)represent the simplest members of the naturally occurring3,4-bisindolylmaleimides. They are structurally related to thearcyriaflavines (b) and to the aglycon of well-known staurosporine (c),rebeccamycine (d) and other biologically active metabolites.

Interestingly, synthetic analogues possess wide spectra ofantibacterial, antiviral, antimicrobial and antigenic activities.Furthermore, derivatives of this class of compounds are promising agentsfor autoimmune diseases, like diabetes and cancer, as well as valuableinhibitors of different protein kinases, especially PKC, which plays animportant role in many signal transduction pathways, or GSK3β,therefore, may be used for the treatment of GSK3β mediated diseases.Notably, some derivatives are currently evaluated in human clinicaltrails as anticancer drugs. For example, Enzastaurin, which is developedby Eli Lilly and Company, is a synthetic bisindolylmaleimide derivativewith potential antineoplastic activity and can be used for the treatmentof solid tumors (WO02/02094, WO02/02116, and IL165747). In January 2009Enzastaurin was in the phase III of the clinical trials. This agent maydecrease tumor blood supply, preventing its growth. Ruboxistaurin,another bisindolylmaleimid, is an investigational drug for diabeticperipheral retinopathy, was also developed by Eli Lilly, and ispresently in a phase III study. Ruboxistaurin is an inhibitor ofPKC-beta. Other examples of indolylmaleimide agents have been describedin WO2009/071620 and WO 2006/061212. Namely, certain 3-(indolyl)- or3-(azaindolyl)-4-arylmaleimide derivatives act as angiogenesisinhibitors therefore were proposed their use for controllingangiogenesis and/or vascular dysfunction as well as for treatment ofcancer.

Obviously, it is very important to develop new strategies to the newderivatives of this class of bioactive compounds that would show moreimproved properties, such as enhanced bioavailability, increasedmetabolic stability, and improved selectivities toward action targetsthat they can be used as targeted drugs.

As a result of pharmaceutical importance of 3,4-bisindolylmaleimides, avariety of approaches have been reported in the literature for theirsynthesis. The most widely used methods were developed by groups of W.Steglich (Tetrahedron, 1988, 44, 2887) and M. Faul (JOC, 1998, 63,6053). Both methods allow the synthesis of symmetrically andunsymmetrically di-substituted maleimides. According to the Steglichprocedure indolyl magnesium bromide reacts with 3,4-dibromomaleimide togive mono- or di-substituted products. The outcome of this reaction isstrongly dependent on the solvent. The procedure of Faul et al. involvesa one step condensation of substituted (aryl or indolyl) acetamides withsubstituted (aryl or indolyl) glyoxyl esters in the presence of strongbase.

Several indolylmaleimide compounds can be also prepared according to theknown methods, which are disclosed, for example in WO02/38561, EP328026,WO03/095452 and WO2006/061212.

Selected compounds of this invention were prepared according to thereference “Org.Biomol.Chem. 2008, 6, 992”. Typically in a two stepsequence first was synthesized 3-halo-4-indolyl- or azaindolylmaleimidederivative, starting from commercially available indole or azaindolederivative and 3,4-dihalomaleimide. In particular case 2-methylindole(1) reacted with 3,4-dibromomaleimide (2) to form3-bromo-1-methyl-4-(2-methyl-3-indolyl)-maleimide (3) (Scheme 2).

Using Grignard reagent according to the protocol of Steglich led to thedesired mono-substituted product in 68% isolated yield. In addition, aminor amount of the corresponding di-substituted product (5%) wasisolated. However, applying the modification of Ohkubo (Tetrahedron,1996, 52, 8099), which means metallation of indole with lithiumhexamethyldisilazane (LiHMDS) and further reaction with one equivalentof dibromo compound 2, led to3-bromo-1-methyl-4-(2-methyl-3-indolyl)-maleimide (3) in excellentselectivity and nearly quantitative yield (98%).

Aryl, heteroaryl or heterocyclyl substituents were introduced in the4-position of maleimide moiety using Suzuki coupling reaction ofcompound 3 with various substituted or non substituted aryl, heteroarylor heterocyclyl boronic acids. The coupling reactions were preferablyperformed in the presence of 0.05 to 4 mol % Pd(OAc)₂ and suitablephosphine ligand. Depend on steric and electronic factors good toexcellent yield of the corresponding product of formula (I) wasobtained. For example, Suzuki coupling reaction of3-bromo-1-methyl-4-(2-methyl-3-indolyl)-maleimide (3) with phenylboronicacid (4) led to1-methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione (5) inquantitative yield (Scheme 3).

All coupling products are bright colored, stabile crystalline compounds.The resulting 3-indolyl-4-aryl(heteroaryl or heterocyclyl)maleimidesconstitute new biologically active compounds. Protection anddeprotection steps of indole nitrogen are not necessary.

As will be apparent to a person skilled in the art, compound of formula(I) wherein X is N—R¹, can be converted to other compound of formula(III) (Scheme 4).

For example, treatment of at maleimide moiety protected compound offormula (I) with strong base, such as sodium or potassium hydroxide ledto the formation of corresponding cyclic anhydrides of formula (II),which are easily converted to unprotected compounds of formula (III)over heating with ammonium acetate.

Both conversions proceed in high to excellent yields. Also theseproducts are bright colored, stabile crystalline compounds.

The present invention is now further illustrated by reference to thefollowing figures and examples from which further advantages, features,and embodiments may be taken, wherein

FIG. 1 shows two diagrams indicating cell number over time when exposedto compound PDA-66 or control (left panel) and viability of cells whenexposed to compound PDA-66 or control (right panel);

FIG. 2 is a bar diagram showing the inhibitory effect of 3 μM PDA-66 ongrowth of SH5Y5 cells, Jurkat cells and B16F10 cells;

FIG. 3 shows a panel of four bar diagrams indicating percentage ofapoptotic cells over time upon exposure to compound PDA-66 with cellsbeing annexin−/PI+, annexin+/PI+, annexin−/PI− or annexin+/PI−;

FIG. 4 shows the percentage of cells in G2/M upon exposure to compoundPDA66;

FIG. 5A is a microphotograph view of cells not exposed to PDA-66;

FIG. 5B is a microphotograph view of cells exposed to PDA-66;

FIG. 5C is a diagram showing optical density over time in apolymerization assay using tubulin upon exposure to various compounds,including PDA-66;

FIG. 6 is a diagram indicating the number of human neuronal progenitorcells (hNPCs), expressed as fold increase, as a function of PDA-66concentration; and

FIG. 7 is a bar diagram showing the inhibitory effect of 3 μM PDA-66(first bar of each experiment) and 10 μM PDA-66 on growth of hNPC cells,B16F10 cells, Jurkat cells, MCF-7 cells, SW480 cells, A 549 cells andHepG2 cells.

EXAMPLES

Abbreviations used in general procedures and examples are defined asfollows: “HCl” for hydrochloric acid, “KOH” for potassium hydroxide,“NaHCO₃” for sodium hydrocarbonate, “K₂CO₃” for potassium carbonate,“Na₂SO₄” for sodium sulfate, “CH₂Cl₂” for methylene chloride, “THF” fortetrahydrofuran, “EA” for ethyl acetate, “DMSO” for dimethylsulfoxide,“CDCl₃” for deuterated chloroform, “TLC” for thin layer chromatography,“LiHMDS” for lithium hexamethyldisilazane, “Pd(OAc)₂” for palladiumacetate.

All reactions were carried out under argon atmosphere. Reactions weremonitored by TLC analysis (pre-coated silica gel plates with fluorescentindicator UV254, 0.2 mm) and visualized with 254 nm UV light or iodine.Chemicals were purchased from Aldrich, Fluka, Acros, AlfaAsar, Strem andunless otherwise noted were used without further purification. Allcompounds were characterized by ¹H NMR, ¹³C NMR, GC-MS, HRMS and IRspectroscopy. ¹H spectra were recorded on Bruker AV 300 and AV 400spectrometers. ¹³C NMR and ¹⁹F NMR spectra were recorded at 75.5 MHz and282 MHz respectively. Chemical shifts are reported in ppm relative tothe center of solvent resonance. Melting points were determined on adigital SMP3 (Stuart). IR spectra were recorded on FT-IR ALPHA (Bruker)with Platinum-ATR (Bruker). EI (70 eV) mass spectra were recorded on MAT95XP (Thermo ELECTRON CORPORATION). GC was performed on Agilent 6890chromatograph with a 30 m HP5 column. HRMS was performed on MAT 95XP(EI) and Agilent 6210 Time-of-Flight LC/MS (ESI). GC-MS was performed onAgilent 5973 chromatograph Mass Selective Detector. All yields reportedrefer to isolated yields.

Example 1: Preparation 1—General Procedure for Condensation of Indole orAzaindoles Derivative with 3,4-dihalomaleimide Compound and SpecificCompounds

The (aza)indole derivative (10 mmol) was dissolved in dry THF (20 ml)and cooled under Argon to −20° C., before 21 ml of LiHMDS (1 M in THF)were slowly added. After stirring for 2 h at −20° C., a solution of3,4-dihalomaleimide derivative (10 mmol) in THF (20 ml) was added to thelithiated (aza)indole solution all at once via syringe. After stirringadditional 1 h at −20° C. (TLC control), the reaction mixture wascarefully neutralized with 2N aq HCl and extracted with ethyl acetate(3×). The combined organics were washed with sat. aq NaHCO₃, brine, andwater. After drying over Na₂SO₄ and concentration, the crude materialwas crystallized from ether.

Example 1.13-Bromo-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (CDCl₃) δ 2.48 (s, 3H), 3.19 (s, 3H), 7.18 (ddd,1H), 7.20 (ddd, 1H), 7.31 (ddd, 1H), 7.48 (m, 1H), 8.48 (br.s, 1H); ¹³CNMR (CDCl₃) δ 14.3, 24.9, 102.0, 110.8, 120.5, 120.7, 120.8, 122.4,126.4, 135.5, 137.6, 139.3, 166.4, 169.1; GC-MS (EI, 70 eV): m/z (%) 318(100) [M^(t)], 320 (96) [M⁺]; HRMS (EI): Cacld for C₁₄H₁₁O₂N₂Br:317.99984. found: 317.99979; IR (ATR, cm⁻¹): 3361, 3066, 1771, 1703,1623, 1422, 1379, 990, 806, 749, 733, 656.

Example 1.23-Bromo-1-methyl-4-(1H-pyrrolo[2,3-b]pyridin-3-yl)-1H-pyrrole-2,5-dione

Preparation was performed using Grignard reagent. Orange crystals; ¹HNMR (DMSO-d₆) δ 2.99 (s, 3H), 7.21 (ddd, 1H, J˜3.83, 5.31, 7.36 Hz),8.20 (s, 1H), 8.31 (dd, 1H, J=1.53, 3.52 Hz), 8.33 (s, 1H), 12.68 (br.s,1H); ¹³C NMR (DMSO-d₆) δ 24.6, 102.7, 114.8, 116.9, 117.0, 130.8, 131.2,136.8, 144.0, 148.7, 166.4, 168.9; GC-MS (EI, 70 eV): m/z (%) 305 (58)[M⁺], 307 (57) [M⁺]; HRMS pos. (ESI): Calc for [M+H]⁺, C₁₂H₉BrN₃O₂:305.98727 and 307.98532; found: 305.98737 and 307.98544; IR (ATR, cm⁻¹):3079, 2742, 1764, 1707, 1584, 1488, 1440, 1419, 1384, 1287, 1167, 1141,1101, 801, 778, 733, 628.

Example 1.31-Methyl-3,4-bis-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (DMSO-d₆) δ 1.97 (s, 3H), 1.98 (s, 3H), 3.05 (s,3H), 6.75 (br. t, 2H, J=7.41 Hz), 6.95 (ddd, 2H, J=3.83, 5.31, 7.36 Hz),7.03 (br. d, 2H, J=7.90 Hz), 7.23 (br. d, 2H, J=8.09 Hz), 11.29 (br.s,2H); ¹³C NMR (DMSO-d₆) δ 13.0, 23.9, 103.3, 110.7, 119.2, 119.4, 120.8,126.6, 131.2, 135.5 (2C), 137.3, 170.4, 171.3; GC-MS (EI, 70 eV): m/z(%) 369 (100) [M⁺]; HRMS (EI): Cacld for C₂₃H₁₉O₂N₃: 369.14718; found369.14705; IR (ATR, cm⁻¹): 3383, 3307, 1755, 1692, 1456, 1435, 1377,1239, 1049, 1022, 1003, 747, 737, 693.

Example 2: Preparation 2—General Procedure for Suzuki Coupling andSpecific Compounds

In an Ace-pressure tube into a solution of (aza)indolylmaleimidederivative (1 mmol) and corresponding boronic acid (1.5 mmol) indimethoxyethane (3 ml) were added K₂CO₃ (1M in water, 3 ml), Pd(OAc)₂ (2mol %) and ligand (2.5 mol %) under argon atmosphere. The pressure tubewas fitted with a Teflon cap and heated at 100° C. (TLC control). Themixture was cooled to room temperature and diluted with ethyl acetate.The organic layer was washed with sat. aq ammonium chloride (2×30 mL)and water. After drying over Na₂SO₄ and removal of the solvent invacuum, the coupling product was isolated by column chromatography inheptane/ethyl acetate.

Example 2.41-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(4-vinylphenyl)-1H-pyrrole-2,5-dione

Red-orange crystals; ¹H NMR (CDCl₃) δ 2.14 (s, 3H), 3.17 (s, 3H), 5.25(dd, 1H, J=0.66, 10.89 Hz), 5.72 (dd, 1H, J=0.70, 17.61 Hz), 6.63 (dd,1H, J=10.88, 17.62 Hz), 6.96 (m, 1H), 7.09 (m, 2H), 7.23 (m, 1H), 7.27(m, 2H), 7.53 (m, 2H), 8.32 (br s, 1H); ¹³C NMR (CDCl₃) δ 13.7, 24.2,103.0, 110.5, 115.0, 120.3, 120.5, 122.0, 126.1 (2C), 126.5, 129.5 (2C),129.6, 132.7, 133.7, 135.7, 136.2, 136.8, 138.1, 171.2, 171.6; GC-MS(EI, 70 eV): m/z (%) 342 (100) [M⁺]; HRMS (EI): Cacld for C₂₂H₁₈O₂N₂:342.13628; found: 342.13618; IR (ATR, cm⁻¹): 3380, 3053, 2920, 1745,1689, 1456, 1428, 1383, 1235, 990, 903, 847, 814, 741, 656.

Example 2.51-Methyl-3-(2-methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (CDCl₃) δ 2.14 (s, 3H), 3.20 (s, 3H), 6.97 (ddd,1H), 7.11 (m, 2H), 7.22 (ddd, 1H), 7.27 (m, 3H), 7.55 (m, 2H), 8.33(br.s, 1H); ¹³C NMR (CDCl₃) δ 13.6, 24.2, 102.8, 110.5, 120.3, 120.5,122.0, 126.5, 128.4 (2C), 129.1, 129.3 (2C), 130.2, 133.2, 134.1, 135.7,136.8, 171.2, 171.5; GC-MS (EI, 70 eV): m/z (%) 316 (100) [M⁺]; HRMS(EI): Cacld for C₂₀H₁₆O₂N₂: 316.12063; found: 316.12091; IR (ATR, cm⁻¹):3426, 3381, 3052, 1759, 1690, 1618, 1435, 1422, 1382, 1234, 1002, 989,938, 786, 752, 736, 693.

Example 2.63-(4-Acetylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione(PDA-66)

Red crystals; ¹H NMR (CDCl₃) δ 2.18 (s, 3H), 2.57 (s, 3H), 3.21 (s, 3H),6.94 (ddd, 1H, J˜0.99, 7.05, 8.00 Hz), 7.03 (ddd, 1H), 7.11 (ddd, 1H,J˜1.15, 7.05, 8.11 Hz), 7.25 (dd, 1H, J˜0.41, 8.11 Hz), 7.67 (ddd, 2H,J˜1.72, 3.63, 8.61 Hz), 7.84 (ddd, 2H, J˜1.85, 3.70, 8.61 Hz), 8.57(br.s, 1H); ¹³C NMR (CDCl₃) δ 13.8, 24.3, 26.6, 102.5, 110.7, 120.1,120.6, 122.2, 126.2, 128.2 (2C), 129.5 (2C), 131.9, 134.9, 135.0, 135.8,136.6, 137.6, 170.8, 171.1, 197.8; GC-MS (EI, 70 eV): m/z (%) 358 (100)[M⁺]; HRMS (EI): Cacld for C₂₂H₁₈O₃N₂: 358.13119; found: 358.131088; IR(ATR, cm⁻¹): 3339, 3058, 2923, 1762, 1692, 1678, 1427, 1407, 1383, 1358,1265, 1234, 990, 846, 817, 742.

Example 2.73-(2,6-Dimethylphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (CDCl₃) δ 1.97 (d, 3H, J=0.88), 2.08 (s, 6H),3.22 (s, 3H), 7.00 (ddd, 1H), 7.01 (d, 2H), 7.10 (ddd, 1H), 7.12 (ddd,1H), 7.19 (ddd, 1H), 7.25 (ddd, 1H), 8.21 (br.s, 1H); ¹³C NMR (CDCl₃) δ13.2, 20.7 (2C), 24.4, 103.6, 110.3, 119.9, 120.6, 122.1, 126.8, 128.0(2C), 128.8, 129.5, 135.4, 136.1, 136.9, 137.0 (2C), 137.1, 171.0,171.2; GC-MS (EI, 70 eV): m/z (%) 344 (100) [M⁺]; HRMS (EI): Cacld forC₂₂H₂₀O₂N₂: 344.15193; found: 344.15175; IR (ATR, cm⁻¹): 3342, 2951,1763, 1689, 1433, 1381, 1229, 987, 739, 665.

Example 2.83-(3-Chlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (CDCl₃) δ 2.20 (s, 3H), 3.20 (s, 3H), 6.98 (ddd,1H, J˜1.03, 7.0, 8.03 Hz), 7.05 (br. d, 1H, J˜7.52 Hz), 7.13 (ddd, 1H,J˜1.23, 7.0, 8.14 Hz), 7.18 (br. t, 1H, J=7.91 Hz), 7.25 (m, 2H), 7.40(ddd, 1H, J˜1.26, 2.72, 7.84 Hz), 7.62 (br. t, 1H, J=1.80 Hz), 8.36(br.s, 1H); ¹³C NMR (CDCl₃) δ 13.8, 24.3, 102.6, 110.6, 120.3, 120.7,122.2, 126.2, 127.5, 129.1, 129.2, 129.6, 131.9, 132.1, 134.2, 134.3,135.85, 137.2, 170.8, 171.1; GC-MS (EI, 70 eV): m/z (%) 350 (100) [M⁺];HRMS (EI): Cacld for C₂₀H₁₅O₂N₂Cl: 350.08166; found: 350.08115; IR (ATR,cm⁻¹): 3350, 3068, 2909, 1764, 1689, 1433, 1383, 1235, 991, 743, 735,715, 683.

Example 2.93-(2,4-Dichlorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (CDCl₃) δ 2.11 (s, 3H), 3.21 (s, 3H), 6.99 (ddd,1H, J˜1.03, 7.0, 8.03 Hz), 7.10 (ddd, 1H, J˜0.93, 7.09, 8.23 Hz), 7.18(m, 2H), 7.19 (d, 2H, J˜1.20 Hz), 7.40 (br. t, 1H, J=1.15 Hz), 8.41(br.s, 1H); ¹³C NMR (CDCl₃) δ 13.5, 24.4, 103.1, 110.6, 119.7, 120.8,122.2, 126.7, 127.3, 128.5, 130.1, 132.1, 132.4, 134.7, 135.5, 137.5,137.6, 170.1, 170.6; GC-MS (EI, 70 eV): m/z (%) 384 (100) [M⁺]; HRMS(EI): Cacld for C₂₀H₁₄O₂N₂Cl₂: 384.04268; found: 384.04261; IR (ATR,cm⁻¹): 3358, 3064, 2949, 1756, 1687, 1436, 1386, 1228, 992, 857, 810,778, 741, 673, 666.

Example 2.101-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(thiophen-3-yl)-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (CDCl₃) δ 2.27 (s, 3H), 3.18 (s, 3H), 7.01 (ddd,1H, J 1.07, 7.07, 8.05 Hz), 7.12 (ddd, 1H), 7.137 (dd, 1H, J˜3.02, 5.15Hz), 7.14 (ddd, 1H), 7.19 (dd, 1H, J=1.22, 5.17 Hz), 7.25 (dt, 1H,J=0.91, 8.06 Hz), 8.11 (dd, 1H, J=1.24, 2.95 Hz), 8.42 (br.s, 1H); ¹³CNMR (CDCl₃) δ 13.5, 24.2, 102.9, 110.6, 120.1, 120.5, 122.0, 125.1,126.7, 127.5, 129.2, 130.0, 130.2. 130.5, 135.7, 136.7, 171.5, 171.6;GC-MS (EI, 70 eV): m/z (%) 322 (100) [M⁺]; HRMS (EI): Cacld forC₁₈H₁₄O₂N₂S: 322.07705; found: 322.07631; IR (ATR, cm⁻¹): 3391, 3102,1756, 1689, 1624, 1438, 1410, 1382, 1334, 1228, 1071, 1003, 989, 820,804, 790, 752, 737, 653.

Example 2.111-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(pyridin-4-yl)-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (CDCl₃) δ 2.28 (s, 3H), 3.21 (s, 3H), 6.97 (m, 2H),7.14 (ddd, 1H, J˜3.58, 4.69, 8.23 Hz), 7.30 (dt, 1H, J˜0.7, 8.15 Hz),7.46 (2dd, 2H, J˜1.59, 4.57 Hz), 8.53 (2dd, 2H, J˜1.57, 4.62 Hz), 8.71(br.s, 1H); ¹³C NMR (CDCl₃) δ 14.1, 24.4, 102.5, 110.8, 120.3, 120.9,122.5, 123.3 (2C), 125.9, 139.9, 135.9, 136.5, 137.9, 138.1, 149.8 (2C),170.3, 170.6; GC-MS (EI, 70 eV): m/z (%) 317 (100) [M⁺]; HRMS (EI):Cacld for C₁₉H₁₅O₂N₃: 317.11588; found: 317.11635; IR (ATR, cm⁻¹): 3342,2923, 1765, 1694, 1456, 1428, 1383, 1237, 990, 813, 742, 656.

Example 2.121-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (CDCl₃) δ 2.09 (s, 3H), 3.24 (s, 3H), 6.95 (ddd,1H, J˜1.04, 7.14, 8.12 Hz), 7.11 (ddd, 1H, J˜1.11, 7.13, 8.18 Hz), 7.20(dd, 1H, J˜0.5, 8.12 Hz), 7.25 (dd, 1H, J˜<0.5, 8.07 Hz), 7.44 (dd, 1H,J˜1.68, 8.58 Hz), 7.48 (m, 2H), 7.59 (br. d, 1H, J˜8.71 Hz), 7.74 (m,1H), 7.83 (m, 1H), 8.33 (br.s, 2H); ¹³C NMR (CDCl₃) δ 13.7, 24.3, 103.1,110.5, 120.4, 120.6, 122.1, 125.8, 126.3, 126.8, 127.1, 127.6, 127.7,127.8, 128.9, 130.0, 133.0, 133.18, 133.22, 133.8, 135.7, 136.9, 171.2,171.6; GC-MS (EI, 70 eV): m/z (%) 366 (100) [M⁺]; HRMS (EI): Cacld forC₂₄H₁₈O₂N₂: 366.13628; found: 366.13581; IR (ATR, cm⁻¹): 3345, 3055,2946, 1759, 1689, 1425, 1381, 1226, 989, 816, 737, 660.

Example 2.133-(2,5-Dimethoxyphenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Deep orange crystals; ¹H NMR (CDCl₃) δ 2.05 (s, 3H), 3.19 (s, 3H), 3.36(s, 3H), 3.66 (s, 3H), 6.75 (br. d, 1H, J˜8.79 Hz), 6.81 (br. d, 1H,J˜2.57 Hz), 6.84 (dd, 1H, J˜3.05, 8.80 Hz), 6.94 (ddd, 1H, J˜1.13, 7.08,8.02 Hz), 7.05 (ddd, 1H, J˜1.07, 7.15, 8.02 Hz), 7.14 (br. d, 1H, J˜8.11Hz), 7.18 (br. d, 1H, J 7.94 Hz), 8.38 (br.s, 1H); ¹³C NMR (CDCl₃) δ13.3, 24.2, 55.78, 55.84, 104.0, 110.2, 112.7, 115.9, 116.2, 119.9,120.3, 120.6, 121.8, 127.0, 133.3, 135.5, 135.6, 136.6, 151.9 (2C),153.4 (2C), 171.0, 171.3; GC-MS (EI, 70 eV): m/z (%) 376 (100) [M⁺];HRMS (EI): Cacld for C₂₂H₂₀O₄N₂: 376.14176; found: 376.14113; IR (ATR,cm⁻¹): 3338, 2924, 1750, 1689, 1427, 1383, 1273, 1237, 1212, 1049, 1018,997, 823, 760, 746, 724, 667.

Example 2.141-Methyl-3-(2-methyl-1H-indol-3-yl)-4-(2-(trifluoromethyl)phenyl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (Aceton-d₆) δ 2.20 (s, 3H), 3.11 (s, 3H), 6.86(ddd, 1H, J˜1.06, 7.13, 8.07 Hz), 7.00 (ddd, 1H, J˜1.16, 7.16, 8.13 Hz),7.19 (br. d, 1H, J˜7.95 Hz), 7.27 (ddd, 1H), 7.37 (m, 1H), 7.55 (m, 2H),7.76 (m, 1H), 10.55 (br.s, 1H); ¹³C NMR (Aceton-d₆) δ 13.1, 24.1, 102.3(d, J=4.55 Hz), 111.2 (d, J=5.13 Hz), 120.0, 120.2, 121.9, 124.9 (q,J=272.93 Hz), 127.7 (q, J=4.42 Hz), 127.9 (d, J=3.68 Hz), 129.6 (q,J=30.37 Hz), 129.9 (d, J=1.79 Hz), 130.0, 132.5 (2C), 135.4, 136.5 (d,J=15.20 Hz), 137.5, 138.4 (d, J=14.52 Hz), 170.87, 170.93; ¹⁹F NMR(CDCl₃) δ−57.57 (s); GC-MS (EI, 70 eV): m/z (%) 384 (100) [M⁺]; HRMS(EI): Cacld for C₂₁H₁₅O₂N₂F₃: 384.10801; found: 384.10765; IR (ATR,cm⁻¹): 3365, 3080, 1768, 1694, 1445, 1385, 1315, 1163, 1118, 1036, 991,764, 742, 657.

Example 2.153-(4-Fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (CDCl₃) δ 2.21 (s, 3H), 3.20 (3, 3H), 6.96 (m,3H), 7.03 (dd, 1H, J˜0.35, 7.75 Hz), 7.12 (ddd, 1H, J˜1.24, 6.93, 8.13Hz), 7.25 (ddd, 1H), 7.59 (ddt, 2H, J˜2.90, 5.50, 8.48 Hz), 8.35 (br.s,1H); ¹³C NMR (CDCl₃) δ 13.7, 24.2, 102.6, 110.6, 115.4, 115.7, 120.2,120.6, 122.1, 126.22 (d, J˜3.60 Hz), 126.3, 131.4, 131.5, 132.87 (d,J˜1.07 Hz), 132.0, 135.8, 136.9, 162.89 (d, J=251.81 Hz), 171.1, 171.5;¹⁹F NMR (CDCl₃) δ−109.8 (s); HRMS (EI): Cacld for C₂₀H₁₅O₂N₂F:334.11121; found: 334.11137; IR (ATR, cm⁻¹): 3380, 3042, 1755, 1700,1600, 1508, 1458, 1427, 1379, 1232, 1159, 996, 841, 813, 750, 731, 657.

Example 2.163-(5-Acetyl-2-fluorophenyl)-1-methyl-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Deep red crystals; ¹H NMR (Aceton-d₆) δ 2.29 (s, 3H), 2.49 (s, 3H), 3.12(s, 3H), 6.80 (ddd, 1H, J˜1.06, 7.15, 8.37 Hz), 7.00 (m, 2H), 7.16 (dd,1H, J=8.69, 9.51 Hz), 7.30 (dd, 1H, J=0.82, 8.69 Hz), 8.01 (ddd, 1H,J=2.34, 4.90, 8.57 Hz), 8.17 (dd, 1H, J=2.23, 6.79 Hz), 10.65 (br.s,1H); ¹³C NMR (Aceton-d₆) δ 13.4, 24.1, 26.3, 103.5, 111.4, 116.6 (d,J=22.4 Hz), 119.9, 120.1 (d, J=16.2 Hz), 120.4, 122.0, 127.4, 128.7 (d,J=2.5 Hz), 131.8 (d, J=9.6 Hz), 133.0 (d, J=4.6 Hz), 134.2 (d, J=3.4Hz), 136.7, 138.4, 138.9, 163.4 (d, J=259.4 Hz), 170.6, 170.8, 195.9;¹⁹F NMR (Aceton-d₆) δ−102.9 (m); GC-MS (EI, 70 eV): m/z (%) 376 (100)[M⁺]; HRMS pos. (ESI): Calc for [M+H]⁺, C₂₂H₁₈FN₂O₃: 377.1296; found:377.1302; HRMS pos. (ESI): Calc for [M+Na]⁺, C₂₂H₁₇FN₂NaO₃: 399.11154;found: 399.11152; IR (ATR, cm⁻¹): 3351, 1689, 1645, 1602, 1439, 1386,1353, 1250, 1223, 828, 778, 742, 630, 568, 436, 408.

Example 2.17N-(4-(1-Methyl-4-(2-methyl-1H-indol-3-yl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)phenyl)acetamide

Orange crystals; ¹H NMR (Aceton-d₆) δ 2.05 (s, 3H), 2.27 (s, 3H), 3.07(s, 3H), 6.83 (ddd, 1H, J˜0.98, 6.93, 8.06 Hz), 7.00 (d, 1H, J=7.60 Hz),7.02 (ddd, 1H), 7.32 (ddd, 1H, J˜1.00, 2.09, 7.76 Hz), 7.53 (m, 4H),9.27 (br.s, 1H), 10.59 (br.s, 1H); ¹³C NMR (Aceton-d₆) δ 13.3, 23.8,24.0, 103.0, 111.3, 118.8 (2C), 120.1, 120.5, 121.8, 125.9, 127.2, 130.6(2C), 132.6, 133.8, 136.9, 137.9, 140.7, 168.8, 171.4, 171.9; GC-MS (EI,70 eV): m/z (%) 373 (100) [M⁺]; HRMS pos. (ESI): Calc for [M+H]⁺,C₂₂H₂₀N₃O₃: 374.14992; found: 374.15012; HRMS pos. (ESI): Calc for[M+Na]⁺, C₂₂H₁₉N₃NaO₃: 396.13186; found: 396.13226; IR (ATR, cm⁻¹):3379, 1675, 1582, 1505, 1424, 1403, 1386, 1365, 1310, 1237, 1179, 851,815, 750, 653, 585, 567, 556, 532, 434, 379.

Example 3: Preparation 3—General Procedure for Preparation of Compoundsof Formula (II) and (III) and Specific Compounds

Step 1. The mixture of compound of formula (I) (1 mmol) wherein X isN—R¹, and 100 ml of 10% aq KOH was heated at 140° C. until the mixturebecome homogenous (10 to 30 min, TLC control). Then the solution wascooled and acidified with 2N aq HCl, until precipitate was formed, whichwas collected, dried and recrystallized to give nearly quantitativelycyclic anhydride of formula (II).

Step 2. Compound of formula (II) (1 mmol) was heated with ammoniumacetate (100 mmol) at 140° C. until the mixture become homogenous (TLCcontrol). The mixture was cooled down, water was added, and the mixturewas extracted with ethyl acetate. The combined organics were washed withwater, dried over Na₂SO₄ and concentrated. The crude material wascrystallized from ether. The product of formula (III) was isolated bycolumn chromatography in heptane/ethyl acetate.

Example 3.18 3-(2-Methyl-1H-indol-3-yl)-4-phenylfuran-2,5-dione

Red crystals; ¹H NMR (Aceton-d₆) δ 2.31 (s, 3H), 6.86 (ddd, 1H, J˜1.03,7.06, 8.08 Hz), 7.02 (ddd, 1H), 7.07 (ddd, 1H, J˜1.14, 7.17, 8.21 Hz),7.36 (m, 4H), 7.60 (m, 2H), 10.83 (br.s, 1H); ¹³C NMR (Aceton-d₆) δ13.4, 102.2, 111.6, 120.6 (2C), 122.4, 126.7, 128.9 (2C), 129.9 (2C),130.2, 130.4, 134.9, 136.1, 136.9, 139.7, 166.1, 166.3; GC-MS (EI, 70eV): m/z (%) 303 (52) [M⁺]; HRMS (EI): Calc for C₁₉H₁₃O₃N: 303.08899;found: 303.08861; IR (ATR, cm⁻¹): 3350, 2921, 2852, 1825, 1749, 1618,1456, 1423, 1252, 902, 741, 726, 693, 671, 635, 622, 564, 531.

Example 3.19 3-(2-Methyl-1H-indol-3-yl)-4-phenyl-1H-pyrrole-2,5-dione

Red crystals; ¹H NMR (Aceton-d₆) δ 2.24 (s, 3H), 6.83 (ddd, 1H, J˜1.01,7.08, 8.01 Hz), 7.02 (ddd, 1H), 7.03 (d, 1H, J=7.58 Hz), 7.27 (m, 3H),7.31 (ddd, 1H), 7.54 (m, 2H), 9.83 (br.s, 1H), 10.56 (br.s, 1H); ¹³C NMR(Aceton-d₆) δ 13.2, 102.8, 111.2, 120.1, 120.5, 121.8, 127.3, 128.6(2C), 129.3, 130.0 (2C), 131.3, 134.8, 134.9, 136.8, 138.0, 171.7,172.2; GC-MS (EI, 70 eV): m/z (%) 302 (100) [M⁺]; HRMS (EI): Calc forC₁₉H₁₄O₂N₂: 302.10498; found: 302.105426; IR (ATR, cm⁻¹): 3379, 3205,3065, 2917, 2764, 1764, 1704, 1598, 1451, 1423, 1335, 1289, 1278, 1227,1013, 993, 770, 754, 729, 719, 690.

Example 3.203-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)furan-2,5-dione

Orange crystals; ¹H NMR (CDCl₃) δ 2.30 (s, 3H), 6.81 (ddd, 1H), 7.05(ddd, 2H), 7.37 (ddd, 1H), 7.53 (m, 3H), 7.75 (d, 1H, J˜8.62 Hz), 7.87(m, 2H), 8.36 (s, 1H), 10.86 (s, 1H); ¹³C NMR (CDCl₃) δ 13.3, 102.5,111.6, 120.6, 120.7, 122.4, 126.0, 126.9, 127.2, 127.7, 128.16, 128.24,128.4, 129.3, 130.7, 133.5, 134.1, 134.7, 136.1, 136.9, 139.9, 166.1,166.4; MS (EI): m/z (%) 353 (650) [M⁺]; HRMS pos. (ESI): Calc for[M+H]⁺, C₂₃H₁₆NO₃: 354.11247; found: 354.11221; HRMS pos. (ESI): Calcfor [M+Na]⁺, C₂₃H₁₅NNaO₃: 376.09441; found: 376.09419; IR (ATR, cm⁻¹):3366, 2926, 1757, 1460, 1428, 1259, 1242, 1222, 1158, 910, 784, 766,737, 590, 554, 475.

Example 3.213-(2-Methyl-1H-indol-3-yl)-4-(naphthalen-2-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (Aceton-d₆) δ 2.24 (s, 3H), 6.77 (ddd, 1H, J1.04, 7.12, 8.07 Hz), 7.00 (ddd, 1H, J˜1.15, 7.10, 8.07 Hz), 7.08 (d,1H, J=8.0 Hz), 7.33 (ddd, 1H, J˜0.85, 8.08), 7.48 (m, 3H), 7.66 (d, 1H,J=8.8 Hz), 7.80 (m, 1H), 7.84 (m, 1H), 8.30 (d, 1H, J=0.72 Hz), 9.90(br.s, 1H), 10.60 (br.s, 1H); ¹³C NMR (Aceton-d₆) δ 13.3, 103.1, 111.3,120.2, 120.5, 121.9, 126.6, 126.9, 127.5 (2C), 128.0, 128.1, 128.9,129.1, 130.4, 133.6, 133.7, 134.5, 135.0, 136.8, 138.3, 171.7, 172.3; MS(EI): m/z (%) 352 (100) [M⁺]; HRMS (EI): Calc for C₂₃H₁₆O₂N₂: 352.12063;found: 352.120553; IR (ATR, cm⁻¹): 3379, 3209, 3062, 2959, 2925, 2738,1762, 1702, 1621, 1457, 1426, 1329, 1290, 1222, 1034, 993, 858, 826,786, 754, 742, 715, 670, 662.

Example 3.223-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)furan-2,5-dione

Red-orange crystals; ¹H NMR (CDCl₃) δ 2.33 (s, 3H), 2.56 (s, 3H), 6.86(ddd, 1H, J˜1.0, 7.04, 8.08 Hz), 6.99 (br. d, 1H, J˜8.0 Hz), 7.07 (ddd,1H, J˜1.22, 7.02, 8.12 Hz), 7.34 (ddd, 1H, J˜0.80, 0.84, 8.10), 7.72(ddd, 2H), 7.94 (ddd, 2H), 10.98 (br.s, 1H); ¹³C NMR (CDCl₃) δ 13.5,26.4, 102.3, 111.7, 120.6, 120.8, 122.5, 126.5, 128.6 (2C), 130.1 (2C),133.3, 134.5, 136.9, 137.5, 138.0, 140.4, 165.9, 166.0, 197.2; GC-MS(EI, 70 eV): m/z (%) 345 (87) [M⁺]; HRMS (EI): Calc for C₂₁H₁₅O₄N:345.09956; found: 345.09942; IR (ATR, cm⁻¹): 3233, 2921, 2852, 1759,1671, 1460, 1252, 1186, 1112, 924, 831, 747, 731, 628, 591, 578, 516,456, 434, 416.

Example 3.23

3-(4-Acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione

Orange crystals; ¹H NMR (Aceton-d₆) δ 2.28 (s, 3H), 2.53 (s, 3H), 6.82(ddd, 1H, J˜1.03, 7.07, 8.03 Hz), 6.99 (br. d, 1H, J˜7.40 Hz), 7.02(ddd, 1H, J˜1.12, 7.07, 8.11 Hz), 7.33 (ddd, 1H, J˜0.81, 0.95, 8.08),7.66 (ddd, 2H), 7.87 (ddd, 2H), 9.93 (br.s, 1H), 10.67 (br.s, 1H); ¹³CNMR (Aceton-d₆) δ 13.4, 26.4, 102.8, 111.4, 120.3, 120.5, 122.0, 127.1,128.4 (2C), 130.2 (2C), 133.3, 135.9, 136.3, 136.9, 137.2, 138.7, 171.4,171.8, 197.2; GC-MS (EI, 70 eV): m/z (%) 344 (100) [M⁺]; HRMS (EI): Calcfor C₂₁H₁₆O₃N₂: 344.11554; found: 344.11495; IR (ATR, cm⁻¹): 3343, 3296,3057, 1757, 1699, 1676, 1428, 1343, 1262, 1230, 740, 666, 638, 595, 460,409.

Example 3.243-(4-Acetylphenyl)-4-(1,2-dimethyl-1H-indol-3-yl)-1-methyl-1H-pyrrole-2,5-dione

Dark red crystals; ¹H NMR (DMSO-d₆) δ 2.18 (s, 3H), 2.52 (s, 3H), 3.03(s, 3H), 3.71 (s, 3H), 6.84 (ddd, 1H), 6.93 (br. d, 1H, J˜7.54 Hz), 7.08(ddd, 1H, J˜1.06, 7.08, 8.14 Hz), 7.45 (br. d, 1H, J˜8.25 Hz), 7.56 (br.d, 2H, J˜8.50 Hz), 7.86 (br. d, 2H, J˜8.50 Hz); ¹³C NMR (DMSO-d₆) δ12.3, 24.2, 26.8, 29.9, 101.3, 109.9, 119.7, 119.9, 121.3, 125.1, 128.1(2C), 129.3 (2C), 131.6, 134.6, 135.1, 136.2, 137.1, 139.6, 170.4,170.7, 197.5; GC-MS (EI, 70 eV): m/z (%) 372 (100) [M⁺]; HRMS pos.(ESI): Calc for [M+H]⁺, C₂₃H₂₁N₂O₃: 373.15467; found: 373.15473; IR(ATR, cm⁻¹): 3433, 2915, 1759, 1680, 1599, 1433, 1404, 1382, 1359, 1265,1240, 957, 849, 829, 749, 738, 726, 595, 546, 439.

Example 3.25 3-(4-Acetylphenyl)-1-methyl-4-(1H-pyrrolo[2,3-b]yridine-3-yl)-1H-pyrrole-2,5-dione

Yellow crystals; ¹H NMR (DMSO-d₆) δ 2.57 (s, 3H), 3.04 (s, 3H), 6.66(dd, 1H, J˜0.89, 8.00 Hz), 6.80 (dd, 1H, J˜4.74, 7.96 Hz), 7.55 (br. d,2H, J˜8.27 Hz), 7.93 (br. d, 2H, J˜8.20 Hz), 8.11 (s, 1H), 8.18 (br. d,2H, J˜3.70 Hz), 12.55 (br.s, 1H); ¹³C NMR (DMSO-d₆) δ 24.1, 26.8, 102.8,116.28, 116.34, 128.0 (2C), 128.4, 129.1, 129.9 (2C), 131.8, 132.5,134.8, 136.5, 143.7, 149.0, 170.6, 170.8, 197.5; GC-MS (EI, 70 eV): m/z(%) 345 (100) [M⁺]; HRMS pos. (ESI): Calc for [M+H]⁺, C₂₀H₁₆N₃O₃:346.11862; found: 346.11828; IR (ATR, cm⁻¹): 3025, 2873, 2817, 1756,1695, 1677, 1440, 1421, 1385, 1289, 1269, 1229, 1090, 814, 776, 750,645, 596, 514.

Example 4: Biological Methods

Human neural progenitor cells ReNcell VM (Millipore, Schwalbach,Germany) were cultured on laminin-coated culture-vessels. Culture mediumconsisted of DMEM/F12 supplemented with B27 media supplement, GlutaMax™,gentamycin and heparin sodium salt and growth factors EGF (epidermalgrowth factor) and bFGF (basic fibroblast growth factor). The humanneuroblastoma cell line SH-SY5Y was obtained from the German ResourceCentre for Biological Material DSMZ (Braunschweig, Germany). Cells werecultivated in DMEM, supplemented with 10% FBS, 1%Penicillin-Streptomycin, 4 mM L-Glutamine. The human T cell leukemiacell line Jurkat was obtained from the German Resource Centre forBiological Material DSMZ (Braunschweig, Germany). Cells were cultivatedin RPMI 1640 medium, supplemented with 10% FBS, 1%Penicillin-Streptomycin. The murine melanoma cell line B16F10 wasobtained from the American Type Culture Collection (ATCC, Manassas,USA), Cells were cultivated in DMEM/GM medium supplemented with 10% FBS,1% Penicillin-Streptomycin. The mammary gland/breast carcinoma cell lineMCF-7 was obtained from American Type Culture Collection (ATCC,Manassas, USA) and cultivated according to standard proceedings inEagle's Minimum Essential Medium (EMEM). The colon carcinoma cell lineSW480 was obtained from American Type Culture Collection (ATCC,Manassas, USA) and cultivated according to standard proceedings inLeibovitz's L15 Medium. The lung carcinoma cell line A549 was obtainedfrom American Type Culture Collection (ATCC, Manassas, USA) andcultivated according to standard proceedings in F-12K Medium (Kaighn'sModification of Ham's F-12 Medium). The liver carcinoma cell line Hep G2was obtained from American Type Culture Collection (ATCC, Manassas, USA)and cultivated according to standard proceedings in Eagle's MinimumEssential Medium (EMEM).

Cell Proliferation and Vitality Assay

Cell proliferation and vitality were measured with a CASY Model TT(Roche, Mannheim, Germany) by electrical current exclusion method. Cellswere seeded in defined numbers into 48-well-plates and proliferated for24 h. Subsequently fresh medium was applied containing compounds orDMSO, in the following referred as control.

VEC-DIC Microscopy of Tubulin Polymerization

In order to evaluate interference with tubulin polymerisation bovineneuronal tubulin was used, diluted in BRB80 buffer (80 mM PIPES, pH 6.8;MgCl2 1 mM; EGTA 1 mM) at a concentration of 2 mg/ml. After addition of0.5 mM GTP and 10% DMSO, initiating tubulin polymerisation, the solutionsamples were incubated at 37° C. in the presence of compound of example6 (PDA-66). After 1 h the samples were analysed by using video enhancedcontrast differential interference contrast (VEC-DIC) microscopy.

Tubulin Polymerisation Turbidity Assay

Purified bovine brain tubulin was used to measure tubulin-polymerisationby determining turbidity in tubulin samples incubated with compound ofexample 6 (PDA-66), paclitaxel or nocodazole (all 1 μM) and 0.6% DMSO inwater as negative control. Optical density (OD) was measured afterinitiation of tubulin assembly at a wavelength of 340 nm continuouslyfor 60 min in lmin intervals at a temperature of 37° C. in a platereader (Tecan, Mainz, Germany).

AnnexinV/PI Apoptosis Detection

To determine the amount of apoptotic and necrotic cells a FITC AnnexinVApoptosis Detection Kit I was used. For FACS analysis, cells weretrypsinised and centrifuged at 100×g at RT for 5 min and washed with PBSwithout Ca2⁺ and Mg²⁺. Annexin V and propidium iodide (PI) were addedaccordingly to the manufacture's instructions. After 15 min incubationat room temperature on a shaker in the dark, the measurement was done byusing FACSCalibur (Becton Dickinson, San Jose, USA) in combination withCell Quest Pro software.

Chemicals mentioned in “Biological Methods” were obtained from:Sigma-Aldrich (Hamburg, Germany): DMSO, PIPES, MgCl2, EGTA, GTP,paclitaxel, nocodazole, PDL, PFA, TritonX-100, primary antibody forα-tubulin. Invitrogen (Karlsruhe, Germany): GlutaMax™, gentamycin,heparin sodium salt DMEM/F12, B27 media supplement. Roche (Mannheim,Germany): EGF (epidermal growth factor), bFGF (basic fibroblast growthfactor. GIBCO (Berlin, Germany): DMEM, L-Glutamine. PAA (Colbe,Germany): FBS. Biochrome (Berlin, Germany): Penicillin-Streptomycin.Cytoskeleton (Denver, USA): bovine neuronal tubulin BK006P. AMSBiotechnology (Frankfurt, Germany): laminin. Molecular Probes(Darmstadt, Germany): Alexa Fluor 568. BD Bioscience (Heidelberg,Germany): FITC AnnexinV Apoptosis Detection Kit I.

Example 5: Biological Data

5.1 Cytotoxic effects were evaluated using human fetal cellsimmortalized by transfection with the oncogen v-myc (Donato et al.,Differential development of neuronal physiological responsiveness in twohuman neural stem cell lines. BMC Neurosci.; 8, 2007), also referred toherein as human neuronal progenitor cells, hNPCs, or ReNcell VM,providing a sensitive screening system and cancer cells, namely SH5Y5,Jurkat and B16F10 cells. Cytotoxic effects were measured by comparisonof cells numbers of DMSO treated cells (control) and cell numbers ofcells treated with compound of above mentioned example 2.6 (PDA66). Cellproliferation and vitality was measured with a CASY Model TT (Roche,Mannheim, Germany) by electrical current exclusion method. Cells wereseeded in defined numbers into 48-well-plates and proliferated for 24 h.Subsequently fresh medium was applied containing compound of example 6(3 μM) (PDA-66) or DMSO, in the following referred as control. Cellnumber and cell vitality was measured after 72 h.

The results are shown in FIG. 1. Cell numbers were measured by thecurrent exclusion method where the number of cells and the vitalitycells was assessed simultaneously. Untreated cells showed an exponentialgrowth over time (FIG. 1A, black dots) where the vitality was stableover time (B black dots). Cells treated with compound of example 2.6(PDA-66) did not proliferate (A black squares) and showed a decreasingvitality (FIG. 1 B, black squares) demonstrating the antiproliferativeffect of compound of example 2.6 (PDA-66).

Compound of example 6 (PDA-66) demonstrated strong significantlyantiproliferative effect on immortalized cells (FIG. 1). In comparisonto control, cells treated with compound of example 2.6 (PDA-66) showedno proliferation (FIG. 1, left panel). After 72 h of treatment cellnumber was even lower than the number of seeded cells. In addition thenumber of vital cells, measured in parallel by the current exclusionmethod was significantly lower in cells treated with compound of example2.6 (PDA-66) (FIG. 1, right panel).

The immortalized cells were also tested to determine whether there is adose-dependent effect of the compound of example 2.6 (PDA-66) on theproliferation of said immortalized cells. The results are shown in FIG.6. As may be taken from FIG. 6 there is a dose-dependent effect of thecompound of example 2.6 (PDA-66) on the proliferation of saidimmortalized cells.

5.2 Furthermore cancer cells were treated with compound of example 2.6(PDA-66) with a concentration of 3 μM for 72 h and cell number wasevaluated using the current exclusion method as described above. Cellnumbers of human neuroblastoma cells (SH5Y5), human T cell leukemia(Jurkat) and murine melanoma cells (B16F10) were reduced or attenuatedsignificantly by the treatment with compound of example 2.6 (PDA-66)demonstrating the anti proliferative effect (FIG. 2).

In a further experiment further cancer cells were treated with compoundof example 2.6 (PDA-66) with a concentration of 3 μM or 10 μM for 72 hand cell number was evaluated using the current exclusion method asdescribed above. Cell numbers of immortalized human neuronal progenitorcells (hNPCs; proliferation relative to control at 3 μM: 3.6±1.2%; andat 10 0.9±0.2%), human T cell leukemia (Jurkat; proliferation relativeto control at 3 μM: 18.2±2.2%; and at 10 31.9±5.9%), murine melanomacells (B16F10; proliferation relative to control at 3 μM: 7.2±1.3%; andat 10 8.3±2.6%), mamma carcinoma cells (MCF-1; proliferation relative tocontrol at 3 μM: 43.0±2.8%; and at 10 38.3±11.8%), colon carcinoma cells(SW480; proliferation relative to control at 3 μM: 61.3±0.6%; and at 1056.6±6.6%), lung carcinoma cells (A 549; proliferation relative tocontrol at 3 μM: 57.1±9.2%; and at 10 μM: 65.7±8.0%) and liver carcinomacells (HepG2; proliferation relative to control at 3 μM: 74.8±6.3%; andat 10 μM: 47.2±3.8%) were evidently reduced or attenuated significantlyafter 72 h by the treatment with compound of example 2.6 (PDA-66)demonstrating the anti-proliferative effect (FIG. 7).

The IC50 value for the compound of example 2.6 (PDA-66), expressed asnM, was 532.3 in case of hNPCs, 1073.0 in case of B16F10 cells, and8981.0 in case of A549 cells.

5.3 It was revealed that the cytoxic effect was most likely based toinduction of apoptosis as an increase of apoptotic cells demonstrated bythe increase of AnnexinV⁺/PI⁻ and AnnexinV⁺/PI⁺ cells upon treatment ofthe cells with compound of example 2.6 (PDA-66) was observed, as may betaken from FIG. 3, right lower and upper panel.

5.4 Induction of apoptosis in proliferative cells, as cancer cells orimmortalized cells, can be based on an arrest of the cells within thecell cycle. Such a mitotic arrest can trigger apoptotic cascades as thecells, e.g., do not pass mitotic check points.

A mitotic arrest of cells in the G2/M phase of cell cycle wasdemonstrated for human immortalized cells treated with compound ofexample 2.6 (PDA-66) (FIG. 4) where the highest amount of cells wasfound after 16 h (68.9±10.3% vs. control 17.4±2.3%). As a measure theincrease in cells with double DNA content was used, where at all pointsin time the amount of treated cells was higher in comparison tountreated cells.

5.5 Mitotic arrest is described to be based on a disturbance of themicrotubule assembly, where the depolymerising potential of indoles isknown (Brancaleand Silvestri, Indole, a core nucleus for potentinhibitors of tubulin polymerization. Med.Res.Rev., 27, 2007).

The ability of compound of example 2.6 (PDA-66) to hinder polymerizationof tubulin was demonstrated by assays employing purified tubulin (FIG.5A, B). Tubulin was treated with the compound of example 2.6 and byusing video enhanced differential interference contrast (VEC-DIC)microscopy the influence of compound of example 6 (PDA-66) onmicrotubule polymerization was proved. Untreated tubulin was able tobuild up MTs (FIG. 5A), i.e. to polymerize, whereas in comparisoncompound of example 2.6 (PDA-66) hindered the MT-polymerization and nobundles or bundle-like structures were observed (FIG. 5B).

The effect of compound of example 2.6 (PDA-66) was also demonstrated ina polymerization assay with purified tubulin using a photometric densitymeasurement (FIG. 5C). Untreated tubulin was used as a control, wherethe measurement over the time resulted in a sigmoid shaped curve. Theobtained curve for paclitaxel possessed a comparable shape but with asteeper slope, typical for microtubule stabilizing agents. In contrastthe application of nocodazol, a microtubule destabilizing agent,resulted in a nearly linear curve, indicating that the polymerization oftubulin was hindered. A comparable result was obtained with tubulintreated with compound of example 2.6 (PDA-66), suggesting adestabilizing action of compound of example 2.6 (PDA-66) on tubulin.Consequently, the maximal OD after 60 min was highest for paclitaxeltreated tubulin (1.18 rel. units) and lowest for nocodazole (1.01 rel.units) and PDA-66 (1.04 rel. units). Accordingly, compound of example2.6 (PDA-66) destabilized tubulin in a way comparable to nocodazole, aknown microtubule destabilizing agent.

The features of the present invention disclosed in the specification,the claims, the sequence listing and/or the drawings may both separatelyand in any combination thereof be material for realizing the inventionin various forms thereof.

1-102. (canceled)
 103. A method of treatment of cancer, wherein themethod comprises administering to a subject in need thereof atherapeutically effective amount of a compound having the followingstructure, or pharmaceutically acceptable salts thereof:

wherein the cancer is selected from the group consisting of breastcancer, liver cancer, mamma carcinoma, lung cancer, colon cancer,gastrointestinal cancer, melanoma, lymphoma, glioma, myeloma, colorectalcancer, neuroblastoma, brain cancer, gastric cancer, and any metastasesof any thereof.
 104. The method of claim 103, wherein the compound is3-(4-acetylphenyl)-4-(2-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione. 105.The method of claim 103, further comprising administering a secondtherapeutic agent, wherein the second therapeutic agent is achemotherapeutic agent.
 106. The method of claim 105, wherein thechemotherapeutic agent is selected from the group consisting ofcytarabine, etoposide, mitoxantron, cyclophosphamide, retinoic acid,daunorubicin, doxorubicin, idarubicin, azacytidine, decitabine, atyrosine-kinase inhibitor, a antineoplastic antibody, vinca alkaloidsand steroids.
 107. The method of claim 106, wherein the chemotherapeuticagent is a tyrosine kinase inhibitor, wherein the tyrosine kinaseinhibitor is selected from the group consisting of sorafenib, dasatinib,nilotinib, nelarabine and fludarabine, or wherein the chemotherapeuticagent is alemtuzumab.
 108. The method of claim 103, wherein the compoundhas an antiproliferative effect on cells of the cancer.